Frangible compounds for pathogen inactivation

ABSTRACT

Compounds and methods for inactivating pathogens in materials are described, including compositions and methods for inactivating pathogens in biological materials such as red blood cell preparations and plasma. The compounds and methods may be used to treat materials intended for in vitro or in vivo use, such as clinical testing or transfusion. The compounds are designed to specifically bind to and react with nucleic acid, and then to degrade to form breakdown products. The degradation reaction is preferably slower than the reaction with nucleic acid.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of application Ser. No. 09/003,115,U.S. Pat. No. 6,093,725 which claims the benefit of U.S. ProvisionalApplication Ser. No. 60/043,696, filed Apr. 15, 1997, and is acontinuation-in-part of U.S. patent application Ser. No. 08/779,885,filed Jan. 6, 1997 abandon; and is a continuation-in-part of U.S. patentapplication Ser. No. 08/779,830, filed Jan. 6, 1997, abandon.

STATEMENT OF RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSOREDRESEARCH

This invention was made with United States government support underGrant 1-RO1-HL53380 from the NHLBI. The United States Government hascertain rights in this invention.

TECHNICAL FIELD

This invention relates to compounds which are useful for inactivatingpathogens in a material, such as a blood product, and to methods of useof the compounds.

BACKGROUND ART

The transmission of disease by blood products and other biologicalmaterials remains a serious health problem. While significant advancesin blood donor screening and blood testing have occurred, viruses suchas hepatitis B (HBV), hepatitis C (HCV), and human immunodeficiencyvirus (HIV) may escape detection in blood products during testing due tolow levels of virus or viral antibodies. In addition to the viralhazard, there are currently no licensed tests to screen for the presenceof bacteria or protozoans in blood intended for use in transfusions. Therisk also exists that a hitherto unknown pathogen may become prevalentin the blood supply and present a threat of disease transmission, as infact occurred before the recognition of the risk of HIV transmission viablood transfusions.

Exposure of laboratory workers to blood or other body fluids alsopresents a health hazard. Twelve thousand health-care workers whose jobsinvolve exposure to blood are infected with hepatitis B virus each year,according to estimates from the Centers for Disease Control (“Guidelinesfor Prevention of Transmission of Human Immunodeficiency Virus andHepatitis B Virus to Health-Care and Public-Safety Workers,” Morbidityand Mortality Weekly Report, vol. 38, no. S-6, June 1989).

Several methods have been proposed to complement donor screening andblood testing to decrease the incidence of disease due to transfusions.The introduction of chemical agents into blood or blood plasma has beensuggested to inactivate pathogens prior to clinical use of the bloodproduct. Nitrogen mustard, CH₃—N(CH₂CH₂Cl)₂, was added to bloodcomponents in an investigation of potential virucidal agents. However,substantial hemolysis occurred at the concentrations necessary toinactivate one of the viruses studied, rendering nitrogen mustardunsuitable for use in blood. LoGrippo et al., Proceedings of the SixthCongress of the International Society of Blood Transfusion, BibliothecaHaematologica (Hollander, ed.), 1958, pp. 225-230.

A “solvent/detergent” (S/D) method for inactivating viruses wasdescribed in Horowitz et al., Blood 79:826 (1992) and in Horowitz etal., Transfusion 25:516 (1985). This method utilized 1%tri(n-butyl)phosphate and 1% Triton X-100 at 30° C. for 4 hours toinactivate viruses in fresh frozen plasma. Piquet et al., Vox Sang.63:251 (1992), used 1% tri(n-butyl)phosphate and 1% Octoxynol-9 toinactivate viruses in fresh frozen plasma. Another method forinactivating viruses in blood involves the addition of phenol orformaldehyde to the blood. U.S. Pat. No. 4,833,165. However, both thesolvent/detergent method and the phenol/formaldehyde method requireremoval of the chemical additives prior to clinical use of the bloodproduct.

Inactivation of pathogens in blood products using photoactivated agentshas also been described; see, e.g., Wagner et al., Transfusion, 34:521(1994). However, due to the absorption of light by hemoglobin in severalregions in the ultraviolet and visible spectrum, phototreatment islimited in its application to materials containing red blood cells.There is also some indication that phototreatment of red blood cellsalters the cells in some manner; see Wagner et al., Transfusion 33:30(1993).

There is thus a need for compositions and methods for treating blood,blood-derived products, and other biological materials, which willinactivate pathogens present in the products or materials withoutrendering the products or materials unsuitable for their intended use.Compositions which do not need to be removed from the biologicalmaterial prior to its use would be particularly useful, as equipment andsupplies needed to remove the compositions would be obviated and thecosts of handling the biological material would be reduced. This placesan additional requirement on the composition, however, in that if thecomposition remains in the biological material, it must not pose ahazard when the biological material is used for its intended purpose.For example, a highly toxic compound which inactivates pathogens in ablood sample would preclude the use of that blood for transfusionpurposes (although the blood sample may still be suitable for laboratoryanalysis).

It is one intention of this invention to provide compositions andmethods of use of the compositions for inactivating pathogens inbiological materials, without rendering the materials unsuitable fortheir intended purpose. Examples of how this may be accomplishedinclude, but are not limited to, using the compounds in an ex vivo or invitro treatment of the biological materials and then removing thecompounds prior to the use of the material; by using a compositionwhich, even though it remains in the material, does not render thematerial unsuitable for its intended use; or by using a compositionwhich, after inactivating pathogens in the material, will break down toproducts, where the breakdown products can remain in the materialwithout rendering the material unsuitable for its intended use.

DISCLOSURE OF THE INVENTION

Accordingly, it is an object of this invention to provide compounds forinactivating pathogens in a material, where such compounds comprise anucleic acid binding moiety; an effector moiety, capable of forming acovalent bond with nucleic acid; and a frangible linker covalentlylinking the nucleic acid moiety and the effector moiety; wherein thefrangible linker degrades so as to no longer covalently link the nucleicacid binding moiety and the effector moiety, under conditions which donot render the material unsuitable for its intended purpose.

It is an additional object of this invention to provide such compoundsfor inactivating pathogens in a material, wherein the nucleic acidbinding moiety is selected from the group consisting of acridine,acridine derivatives, psoralen, isopsoralen and psoralen derivatives.

It is an additional object of this invention to provide such compoundsfor inactivating pathogens in a material, wherein the frangible linkercomprises a functional unit selected from the group consisting offorward esters, reverse esters, forward amides, reverse amides, forwardthioesters, reverse thioesters, forward and reverse thionoesters,forward and reverse dithioic acids, sulfates, forward and reversesulfonates, phosphates, and forward and reverse phosphonate groups, asdefined herein.

It is an additional object of this invention to provide such compoundsfor inactivating pathogens in a material, wherein the effector groupcomprises a functional unit which is an alkylating agent.

It is an additional object of this invention to provide such compoundsfor inactivating pathogens in a material, wherein the effector groupcomprises a functional unit selected from the group consisting ofmustard groups, mustard group equivalents, epoxides, aldehydes, andformaldehyde synthons.

It is an additional object of this invention to provide compounds of theformula:

wherein at least one of R₁, R₂, R₃, R₄, R₅, R₆, R₇, R₈ and R₉ is—V—W—X—E as defined below, and the remainder of R₁, R₂, R₃, R₄, R₅, R₆,R₇, R₈ and R₉ are independently selected from the group consisting of—H, —R₁₀, —O—R₁₀, —NO₂, —NH₂, —NH—R₁₀, —N(R₁₀)₂, —F, —Cl, —Br, —I,—C(═O)—R₁₀, —C(═O)—O—R₁₀, and —O—C(═O)—R₁₀,

where —R₁₀ is independently H, —C₁₋₈alkyl, —C₁₋₈heteroalkyl, -aryl,-heteroaryl, —C₁₋₃alkyl-aryl, —C₁₋₃heteroalkyl-aryl,—C₁₋₃alkyl-heteroaryl, —C₁₋₃heteroalkyl-heteroaryl, -aryl-C₁₋₃alkyl,-aryl-C₁₋₃heteroalkyl, -heteroaryl-C₁₋₃alkyl,-heteroaryl-C₁₋₃heteroalkyl, —C₁₋₃alkyl-aryl-C₁₋₃alkyl,—C₁₋₃heteroalkyl-aryl-C₁₋₃alkyl, —C₁₋₃alkyl-heteroaryl-C₁₋₃alkyl,—C₁₋₃alkyl-aryl-C₁₋₃heteroalkyl, —C₁₋₃heteroalkyl-heteroaryl-C₁₋₃alkyl,—C₁₋₃heteroalkyl-aryl-C₁₋₃heteroalkyl,—C₁₋₃alkyl-heteroaryl-C₁₋₃heteroalkyl, or—C₁₋₃heteroalkyl-heteroaryl-C₁₋₃heteroalkyl;

V is independently —R₁₁—, —NH—R₁₁— or —N(CH₃)—R₁₁—, where —R₁₁— isindependently —C₁₋₈alkyl-, —C₁₋₈heteroalkyl-, -aryl-, -heteroaryl-,—C₁₋₃alkyl-aryl-, —C₁₋₃heteroalkyl-aryl-, —C₁₋₃alkyl-heteroaryl-,—C₁₋₃heteroalkyl-heteroaryl-, -aryl-C₁₋₃alkyl-, -aryl-C₁₋₃heteroalkyl-,-heteroaryl-C₁₋₃alkyl-, -heteroaryl-C₁₋₃heteroalkyl-,—C₁₋₃alkyl-aryl-C₁₋₃alkyl-, —C₁₋₃heteroalkyl-aryl-C₁₋₃alkyl-,—C₁₋₃alkyl-heteroaryl-C₁₋₃alkyl-, —C₁₋₃alkyl-aryl-C₁₋₃heteroalkyl-,—C₁₋₃heteroalkyl-heteroaryl-C₁₋₃alkyl-,—C₁₋₃heteroalkyl-aryl-C₁₋₃heteroalkyl-,—C₁₋₃alkyl-heteroaryl-C₁₋₃heteroalkyl-, or—C₁₋₃heteroalkyl-heteroaryl-C₁₋₃heteroalkyl-;

W is independently —C(═O)—O—, —O—C(═O)—, —C(═S)—O—, —O—C(═S)—,—C(═S)—S—, —S—C(═S)—, —C(═O)—S—C(═O)—, —O—S(═O)₂—O—, —S(═O)₂—O—,—S(═O)₂—, —C(═O)—NR₁₀—, —NR₁₀—C(═O)—, —O—P(═O)(—OR₁₀)—O—,—P(═O)(—OR₁₀)—O—, —O—P(═O)(—OR₁₀)—;

X is independently —R₁₁—; and

E is independently selected from the group consisting of —N(R₁₂)₂,—N(R₁₂)(R₁₃), —S—R₁₂, and

 where —R₁₂ is —CH₂CH₂—G, where each G is independently —Cl, —Br, —I,—O—S(═O)₂—CH₃, —O—S(═O)₂—CH₂—C₆H₅, or —O—S(═O)₂—C₆H₄—CH₃;

and where R₁₃ is independently —C₁₋₈alkyl, —C₁₋₈heteroalkyl, -aryl,-heteroaryl, —C₁₋₃alkyl-aryl, —C₁₋₃heteroalkyl-aryl,—C₁₋₃alkyl-heteroaryl, —C₁₋₃heteroalkyl-heteroaryl, —aryl-C₁₋₃alkyl,-aryl-C₁₋₃heteroalkyl, -heteroaryl-C₁₋₃alkyl,-heteroaryl-C₁₋₃heteroalkyl, —C₁₋₃alkyl-aryl-C₁₋₃alkyl,—C₁₋₃heteroalkyl-aryl-C₁₋₃alkyl, —C₁₋₃alkyl-heteroaryl-C₁₋₃alkyl,—C₁₋₃alkyl-aryl-C₁₋₃heteroalkyl, —C₁₋₃heteroalkyl-heteroaryl-C₁₋₃alkyl,—C₁₋₃heteroalkyl-aryl-C₁₋₃heteroalkyl,—C₁₋₃alkyl-heteroaryl-C₁₋₃heteroalkyl, or—C₁₋₃heteroalkyl-heteroaryl-C₁₋₃heteroalkyl;

and all salts and stereoisomers (including enantiomers anddiastereomers) thereof.

It is another object of this invention to provide compounds of theformula:

where R₁, R₂, R₃, R₄, R₅, R₆, R₇, and R₈ are independently selected fromthe group consisting of —H, —R₁₀, —O—R₁₀, —NO₂, —NH₂, —NH—R₁₀, —N(R₁₀)₂,—F, —Cl, —Br, —I, —C(═O)—R₁₀, —C(═O)—O—R₁₀, and —O—C(═O)—R₁₀,

where —R₁₀ is independently H, —C₁₋₈alkyl, —C₁₋₈heteroalkyl, -aryl,-heteroaryl, —C₁₋₃alkyl-aryl, —C₁₋₃heteroalkyl-aryl,—C₁₋₃alkyl-heteroaryl, —C₁₋₃heteroalkyl-heteroaryl, -aryl-C₁₋₃alkyl,-aryl-C₁₋₃heteroalkyl, -heteroaryl-C₁₋₃alkyl,-heteroaryl-C₁₋₃heteroalkyl, —C₁₋₃alkyl-aryl-C₁₋₃alkyl,—C₁₋₃heteroalkyl-aryl-C₁₋₃alkyl, —C₁₋₃alkyl-heteroaryl-C₁₋₃alkyl,—C₁₋₃alkyl-aryl-C₁₋₃heteroalkyl, —C₁₋₃heteroalkyl-heteroaryl-C₁₋₃alkyl,—C₁₋₃heteroalkyl-aryl-C₁₋₃heteroalkyl,—C₁₋₃alkyl-heteroaryl-C₁₋₃heteroalkyl, or—C₁₋₃heteroalkyl-heteroaryl-C₁₋₃heteroalkyl;

R₂₀ is —H or —CH₃; and

R₂₁, is —R₁₁—W—X—E,

where —R₁₁— is independently —C₁₋₈alkyl-, —C₁₋₈heteroalkyl-, -aryl-,-heteroaryl-, —C₁₋₃alkyl-aryl-, —C₁₋₃heteroalkyl-aryl-,—C₁₋₃alkyl-heteroaryl-, —C₃heteroalkyl-heteroaryl-, -aryl-C₁₋₃alkyl-,-aryl-C₁₋₃heteroalkyl-, -heteroaryl-C₁₋₃alkyl-,-heteroaryl-C₁₋₃heteroalkyl-, —C₁₋₃alkyl-aryl-C₁₋₃alkyl-,—C₁₋₃heteroalkyl-aryl-C₁₋₃alkyl-, —C₁₋₃alkyl-heteroalkyl-,—C₁₋₃alkyl-aryl-C₁₋₃heteroalkyl-,—C₁₋₃heteroalkyl-heteroaryl-C₁₋₃alkyl-,—C₁₋₃heteroalkyl-aryl-C₁₋₃heteroalkyl-,—C₁₋₃alkyl-heteroaryl-C₁₋₃heteroalkyl-, or—C₁₋₃heteroalkyl-heteroaryl-C₁₋₃heteroalkyl-;

W is independently —C(═O)—O—, —O—C(═O)—, —C(═S)—O—, —O—C(═S)—,—C(═S)—S—, —S—C(═S)—, —C(═O)—S—C(═O)—, —O—S(═O)₂—O—, —S(═O)₂—O—,—S(═O)₂—, —C(═O)—NR₁₀—, —NR₁₀—C(═O)—, —O—P(═O)(—OR₁₀)—O—,—P(═O)(—OR₁₀)—O—, —O—P(═O)(—OR₁₀)—;

X is independently —R₁₁—; and

E is independently selected from the group consisting of —N(R₁₂)₂,—N(R₁₂)(R₁₃), —S—R₁₂, and

where —R₁₂ is —CH₂CH₂—G, where each G is independently —Cl, —Br, —I,—O—S(═O)₂—CH₃, —O—S(═O)₂—CH₂—C₆H₅, or —O—S(═O)₂—C₆H₄—CH₃;

and where R₁₃ is independently —C₁₋₈alkyl, —C₁₋₈heteroalkyl, -aryl,-heteroaryl, —C₁₋₃alkyl-aryl, —C₁₋₃heteroalkyl-aryl,—C₁₋₃alkyl-heteroaryl, —C₁₋₃heteroalkyl-heteroaryl, -aryl-C₁₋₃alkyl,-aryl-C₁₋₃heteroalkyl, -heteroaryl-C₁₋₃alkyl,-heteroaryl-C₁₋₃heteroalkyl, —C₁₋₃alkyl-aryl-C₁₋₃alkyl,—C₁₋₃heteroalkyl-aryl-C₁₋₃alkyl, —C₁₋₃alkyl-heteroaryl-C₁₋₃alkyl,—C₁₋₃alkyl-aryl-C₁₋₃heteroalkyl, —C₁₋₃heteroalkyl-heteroaryl-C₁₋₃alkyl,—C₁₋₃heteroalkyl-aryl-C₁₋₃heteroalkyl,—C₁₋₃alkyl-heteroaryl-C₁₋₃heteroalkyl, or—C₁₋₃heteroalkyl-heteroaryl-C₁₋₃heteroalkyl;

and all salts and stereoisomers (including enantiomers anddiastereomers) thereof.

It is another object of this invention to provide compounds of theformula:

wherein at least one of R₄₄, R₅₅, R₃, R₄, R₅ and R₈ is —V—W—X—E, and theremainder of R₄₄, R₅₅, R₃, R₄, R₅, and R₈ are independently selectedfrom the group consisting of —H, —R₁₀, —O—R₁₀, —NO₂, —NH₂, —NH—R₁₀,—N(R₁₀)₂, —F, —Cl, —Br, —I, —C(═O)—R₁₀, —C(═O)—O—R₁₀, and —O—C(═O)—R₁₀,

where —R₁₀ is independently H, —C₁₋₈alkyl, —C₁₋₈heteroalkyl, -aryl,-heteroaryl, —C₁₋₃alkyl-aryl, —C₁₋₃heteroalkyl-aryl,—C₁₋₃alkyl-heteroaryl, —C₁₋₃heteroalkyl-heteroaryl, -aryl-C₁₋₃alkyl,-aryl-C₁₋₃heteroalkyl, -heteroaryl-C₁₋₃alkyl,-heteroaryl-C₁₋₃heteroalkyl, —C₁₋₃alkyl-aryl-C₁₋₃alkyl,—C₁₋₃heteroalkyl-aryl-C₁₋₃alkyl, —C₁₋₃alkyl-heteroaryl-C₁₋₃alkyl,—C₁₋₃alkyl-aryl-C₁₋₃heteroalkyl, —C₁₋₃heteroalkyl-heteroaryl-C₁₋₃alkyl,—C₁₋₃heteroalkyl-aryl-C₁₋₃heteroalkyl,—C₁₋₃alkyl-heteroaryl-C₁₋₃heteroalkyl, or—C₁₋₃heteroalkyl-heteroaryl-C₁₋₃heteroalkyl;

V is independently —R₁₁—, —NH—R₁₁— or —N(CH₃)—R₁₁—, where —R₁₁— isindependently —C₁₋₈alkyl-, —C₁₋₈heteroalkyl-, -aryl-, -heteroaryl-,—C₁₋₃alkyl-aryl-, —C₁₋₃heteroalkyl-aryl-, —C₁₋₃alkyl-heteroaryl-,—C₁₋₃heteroalkyl-heteroaryl-, -aryl-C₁₋₃alkyl-, -aryl-C₁₋₃heteroalkyl-,-heteroaryl-C₁₋₃alkyl-, -heteroaryl-C₁₋₃heteroalkyl-,—C₁₋₃alkyl-aryl-C₁₋₃alkyl-, —C₁₋₃heteroalkyl-aryl-C₁₋₃alkyl-,—C₁₋₃alkyl-heteroaryl-C₁₋₃alkyl-, —C₁₋₃alkyl-aryl-C₁₋₃heteroalkyl-,—C₁₋₃heteroalkyl-heteroaryl-C₁₋₃alkyl-,—C₁₋₃heteroalkyl-aryl-C₁₋₃heteroalkyl-,—C₁₋₃alkyl-heteroaryl-C₁₋₃heteroalkyl-, or—C₁₋₃heteroalkyl-heteroaryl-C₁₋₃heteroalkyl-;

W is independently —C(═O)—O—, —O—C(═O)—, —C(═S)—O—, —O—C(═S)—,—C(═S)—S—, —S—C(═S)—, —C(═O)—, —S—C(═O)—, —O—S(═O)₂—O—, —S(═O)₂—O—,—O—S(═O)₂—, —C(═O)—NR₁₀—, —NR₁₀—C(═O)—, —O—P(═O)(—OR₁₀)—O—,—P(═O)(—OR₁₀)—O—, —O—P(═O)(—OR₁₀)—;

X is independently —R₁₁—, and

E is independently selected from the group consisting of —N(R₁₂)₂,—N(R₁₂)(R₁₃), —S—R₁₂, and

 where —R₁₂ is —CH₂CH₂—G, where each G is independently —Cl, —Br, —I,—O—S(═O)₂—CH₃, —O—S(═O)₂—CH₂, —C₆H₅, or —O—S(═O)₂—C₆H₄—CH₃;

and where R₁₃ is independently —C₁₋₈alkyl, —C₁₋₈heteroalkyl, -aryl,-heteroaryl, —C₁₋₃alkyl-aryl, —C₁₋₃heteroalkyl-aryl,—C₁₋₃alkyl-heteroaryl, —C₁₋₃heteroalkyl-heteroaryl, -aryl-C₁₋₃alkyl,-aryl-C₁₋₃heteroalkyl, -heteroaryl-C₁₋₃alkyl,-heteroaryl-C₁₋₃heteroalkyl, —C₁₋₃alkyl-aryl-C₁₋₃alkyl,—C₁₋₃heteroalkyl-aryl-C₁₋₃alkyl, —C₁₋₃alkyl-heteroaryl-C₁₋₃alkyl,—C₁₋₃alkyl-aryl-C₁₋₃heteroalkyl, —C₁₋₃heteroalkyl-heteroaryl-C₁₋₃alkyl,—C₁₋₃heteroalkyl-aryl-C₁₋₃heteroalkyl,—C₃alkyl-heteroaryl-C₁₋₃heteroalkyl, or—C₁₋₃heteroalkyl-heteroaryl-C₁₋₃heteroalkyl;

and all salts and stereoisomers (including enantiomers anddiastereomers) thereof.

It is yet another object of this invention to provide the compoundsβ-alanine, N-(2-carbomethoxyacridin-9-yl),2-[bis(2-chloroethyl)amino]ethyl ester; 4-aminobutyric acidN[(2-carbomethoxyacridin-]-yl), 2-[bis(2-chloroethyl)amino]ethyl ester;5-aminovaleric acid N-[(2-carbomethoxyacridin-9-yl),2-[bis(2-chloroethyl)amino]ethyl ester; β-alanine,N-(2-carbomethoxyacridin-]-yl), 3-[bis(2-chloroethyl)amino]propyl ester;β-alanine, [N,N-bis(2-chloroethyl)],3-[(6-chloro-2-methoxyacridin-9-yl)amino]propyl ester; β-alanine,[N,N-bis(2-chloroethyl)], 2-[(6-chloro-2-methoxyacridin-9-yl)amino]ethylester; [N,N-bis(2-chloroethyl)]-2-aminoethyl4,5′,8-trimethyl-4′-psoralenacetate; and β-alanine, N-(acridin-9-yl),2-[bis(2-chloroethyl)amino]ethyl ester; and all salts thereof.

Provided are methods for inactivating pathogens in a material, such as abiological material, the methods comprising adding one or more compoundsof the invention to the material; and incubating the material. Thecompound may be added to the material to form a final solution having aconcentration of the compound (or total concentration of all compounds,if more than one is used), for example, of between 1 and 500 μM.Biological materials which may be treated include blood, blood products,plasma, platelet preparations, red blood cells, packed red blood cells,serum, cerebrospinal fluid, saliva, urine, sweat, feces, semen, milk,tissue samples, and homogenized tissue samples, derived from human orother mammalian or vertebrate sources.

BEST MODE FOR CARRYING OUT THE INVENTION

This invention provides for compounds useful for inactivating pathogensfound in materials, particularly for inactivating pathogens found inbiological materials such as blood or other body fluids. This inventionalso provides for methods of use of such compounds for inactivatingpathogens in materials. The invention also provides for inactivatingpathogens found in or on materials for biological use. The compounds maybe used in vitro and ex vivo. The biological materials or materials forbiological use may be intended for use in vitro, in vivo, or ex vivo.

The compounds are designed to inactivate pathogens by reacting withnucleic acid. In aqueous solution, at appropriate pH values, thecompounds have a period of activity during which they can bind to andreact with nucleic acid. After this period, the compounds break down toproducts which are no longer able to bind to nor react with nucleicacid.

The chemical organization of the compounds can be broadly described asan anchor, covalently bonded to a frangible linker, which is covalentlybonded to an effector. “Anchor” is defined as a moiety which bindsnon-covalently to a nucleic acid biopolymer (DNA or RNA). “Effector” isdefined as a moiety which reacts with nucleic acid by a mechanism whichforms a covalent bond with the nucleic acid. “Frangible linker” isdefined as a moiety which serves to covalently link the anchor andeffector, and which will degrade under certain conditions so that theanchor and effector are no longer linked covalently. Theanchor-frangible linker-effector arrangement enables the compounds tobind specifically to nucleic acid (due to the anchor's binding ability).This brings the effector into proximity for reaction with the nucleicacid.

The compounds are useful for inactivating pathogens found in materials,particularly biological materials such as blood and other body fluids.Intracellular and extracellular and or other pathogen materials may beinactivated. For example, when a compound of the invention is combinedwith a pathogen-containing red blood cell composition at physiologicalpH, the effector portion of the compound reacts with pathogen nucleicacid. Effector moieties which do not react with nucleic acid aregradually hydrolyzed by the solvent. Hydrolysis of the frangible linkeroccurs concurrently with the effector-nucleic acid reaction and effectorhydrolysis. It is desirable that the frangible linker break down at arate slow enough to permit inactivation of pathogens in the material;that is, the rate of breakdown of the frangible linker is slower thanthe rate at which the compound reacts with nucleic acid. After asufficient amount of time has passed, the compound has broken down intothe anchor (which may also bear fragments of the frangible linker) andthe effector-nucleic acid breakdown products (where fragments of thefrangible linker may also remain attached to the effector), or into theanchor (which may also bear fragments of the frangible linker) and thehydrolyzed effector breakdown products (where fragments of the frangiblelinker may also remain attached to the effector). Additional fragmentsof the frangible linker may also be generated upon degradation of thecompound which do not remain bonded to either the anchor or theeffector. The exact embodiment of the compound of the inventiondetermines whether the anchor breakdown product or the effectorbreakdown product bears fragments of the frangible linker, or whetheradditional fragments of the frangible linker are generated which do notremain bonded to either the anchor or the effector breakdown products.

A preferred embodiment of the invention comprises compounds which, uponcleavage of the frangible linker, result in breakdown products of lowmutagenicity. Mutagenicity of the compounds, after hydrolysis of theeffector, is due primarily to the anchor moiety, as the anchor interactswith nucleic acid and may have the potential to interfere with nucleicacid replication, even if the effector moiety has been hydrolyzed.Preferably, after cleavage of the frangible linker, the anchor fragmenthas substantially reduced mutagenicity.

Definitions

“Pathogen” is defined as any nucleic acid containing agent capable ofcausing disease in a human, other mammals, or vertebrates. Examplesinclude microorganisms such as unicellular or multicellularmicroorganisms. Examples of pathogens are bacteria, viruses, protozoa,fungi, yeasts, molds, and mycoplasmas which cause disease in humans,other mammals, or vertebrates. The genetic material of the pathogen maybe DNA or RNA, and the genetic material may be present assingle-stranded or double-stranded nucleic acid. The nucleic acid of thepathogen may be in solution, intracellular, extracellular, or bound tocells. Table I lists examples of viruses, and is not intended to limitthe invention in any manner.

TABLE I Family: Virus: Adeno Adenovirus 2 Canine hepatitis ArenaPichinde Lassa Bunya Turlock California encephalitis Herpes Herpessimplex 1 Herpes simplex 2 Cytomegalovirus Pseudorabies OrothomyxoInfluenza Papova SV-40 Paramyxo Measles Mumps Parainfluenza 2 and 3Picorna Poliovirus 1 and 2 Coxsackie A-9 Echo 11 Pox Vaccinia Fowl PoxReo Blue tongue Colorado tick fever Retro HIV Avian sarcoma Murinesarcoma Murine leukemia Rhabdo Vesicular stomatitis virus Toga Westernequine encephalitis Dengue 2 Dengue 4 St. Louis encephalitis Hepadnahepatitis B Bacteriophage Lambda T2 (Rickettsia) R. akari(rickettsialpox)

“In vivo” use of a material or compound is defined as introduction ofthe material or compound into a living human, mammal, or vertebrate.

“In vitro” use of a material or compound is defined as a use of thematerial or compound outside a living human, mammal, or vertebrate,where neither the material nor compound is intended for reintroductioninto a living human, mammal, or vertebrate. An example of an in vitrouse would be the analysis of components of a blood sample usinglaboratory equipment.

“Ex vivo” use of a compound is defined as using a compound for treatmentof a biological material outside a living human, mammal, or vertebrate,where that treated biological material is intended for use inside aliving human, mammal, or vertebrate. For example, removal of blood froma human, and introduction of a compound into that blood to inactivatepathogens, is defined as an ex vivo use of that compound if the blood isintended for reintroduction into that human or another human.Reintroduction of the human blood into that human or another human wouldbe in vivo use of the blood, as opposed to the ex vivo use of thecompound. If the compound is still present in the blood when it isreintroduced into the human, then the compound, in addition to its exvivo use, is also introduced in vivo.

“Biological material” is defined as a material originating from abiological organism of any type. Examples of biological materialsinclude, but are not limited to, blood, blood products such as plasma,platelet preparations, red blood cells, packed red blood cells, andserum, cerebrospinal fluid, saliva, urine, feces, semen, sweat, milk,tissue samples, homogenized tissue samples, and any other substancehaving its origin in a biological organism. Biological materials alsoinclude synthetic material incorporating a substance having its originin a biological organism, such as a vaccine preparation comprised ofalum and a pathogen (the pathogen, in this case, being the substancehaving its origin in a biological organism), a sample prepared foranalysis which is a mixture of blood and analytical reagents, cellculture medium, cell cultures, viral cultures, and other culturesderived from a living organism.

“Material for biological use” is defined as any material that will comeinto contact with, or be introduced into, a living human, mammal, orvertebrate, where such contact carries a risk of transmitting disease orpathogens. Such materials include, but are not limited to, medicalimplants such as pacemakers and artificial joints; implants designed forsustained drug release; needles, intravenous lines, and the like; dentaltools; dental materials such as tooth crowns; catheters; and any othermaterial which, when in contact with or introduced into a living human,mammal, or vertebrate, entails risk of transmitting disease orpathogens.

“Inactivation of pathogens” is defined as rendering pathogens in amaterial incapable of reproducing. Inactivation is expressed as thenegative logarithm of the fraction of remaining pathogens capable ofreproducing. Thus, if a compound at a certain concentration renders 99%of the pathogens in a material incapable of reproduction, 1% or one-onehundredth (0.01) of the pathogens remain capable of reproduction. Thenegative logarithm of 0.01 is 2, and that concentration of that compoundis said to have inactivated the pathogens present by 2 logs.Alternatively, the compound is said to have 2 logs kill at thatconcentration.

“Alkyl” as used herein refers to a cyclic, branched, or straight chainchemical group containing carbon and hydrogen, such as methyl, pentyl,and adamantyl. Alkyl groups can either be unsubstituted or substitutedwith one or more substituents, e.g., halogen, alkoxy, acyloxy, amino,hydroxyl, thiol, carboxy, benzyloxy, phenyl, benzyl, or otherfunctionality. Alkyl groups can be saturated or unsaturated (e.g.,containing —C═C— or —C≡—C— subunits), at one or several positions.Typically, alkyl groups will comprise 1 to 12 carbon atoms, preferably 1to 10 carbon atoms, and more preferably 1 to 8 carbon atoms, unlessotherwise specified.

“Heteroalkyl” as used herein are alkyl chains with one or more N, O, S,or P heteroatoms incorporated into the chain. The heteroatom(s) may bearnone, one, or more than one of the substituents described above.“Heteroatoms” also includes oxidized forms of the heteroatoms N, S andP. Examples of heteroalkyl groups include (but are not limited to)methoxy, ethoxy, and other alkyloxy groups; ether-containing groups;amide containing groups such as polypeptide chains; ring systems such aspiperidinyl, lactam and lactone; and other groups which incorporateheteroatoms into the carbon chain. Typically, heteroalkyl groups willcomprise, in addition to the heteroatom(s), 1 to 12 carbon atoms,preferably 1 to 10 carbon atoms, and more preferably 1 to 8 carbonatoms, unless otherwise specified.

“Aryl” or “Ar” refers to an unsaturated aromatic carbocyclic grouphaving a single ring (e.g., phenyl) or multiple condensed rings (e.g.,naphthyl or anthryl), which can be optionally unsubstituted orsubstituted with amino, hydroxyl, C₁₋₈alkyl, alkoxy, halo, thiol, andother substituents.

“Heteroaryl” groups are unsaturated aromatic carbocyclic groups having asingle ring (e.g., pyridyl or furyl) or multiple condensed rings (e.g.,acridinyl, indolyl or benzothienyl) and having at least one hetero atom,such as N, O, or S, within at least one of the rings. The ring(s) canoptionally be unsubstituted or substituted with amino, hydroxyl, alkyl,alkoxy, halo, thiol, acyloxy, carboxy, benzyloxy, phenyl, benzyl, andother substituents.

Abbreviations

The following abbreviations are used: QM (quinacrine mustard); Hct(hematocrit); RBC (red blood cell); LB (Luria Broth); cfu (colonyforming units); pfu (plaque forming units); DMEM (Delbecco's modifiedeagles medium); FBS (fetal bovine serum); PRBC (packed red blood cells);rpm (revolutions per minute); TC (tissue culture); NHSP (normal humanserum pool); NCS (newborn calf serum); PBS (phosphate buffered saline).

Chemical Structure of the Compounds

A wide variety of groups are available for use as the anchors, linkers,and effectors. Examples of anchor groups which can be used in thecompound include, but are not limited to, intercalators, minor groovebinders, major groove binders, molecules which bind by electrostaticinteractions such as polyamines, and molecules which bind by sequencespecific interactions. The following is a non-limiting list of possibleanchor groups:

acridines (and acridine derivatives, e.g. proflavine, acriflavine,diacridines, acridones, benzacridines, quinacrines), actinomycins,anthracyclinones, rhodomycins, daunomycin, thioxanthenones (andthioxanthenone derivatives, e.g. miracil D), anthramycin, mitomycins,echinomycin (quinomycin A), triostins, ellipticine (and dimers, trimersand analogs thereof), norphilin A, fluorenes (and derivatives, e.g.flourenones, fluorenodiamines), phenazines, phenanthridines,phenothiazines (e.g., chlorpromazine), phenoxazines, benzothiazoles,xanthenes and thioxanthenes, anthraquinones, anthrapyrazoles,benzothiopyranoindoles, 3,4-benzopyrene, 1-pyrenyloxirane,benzanthracenes, benzodipyrones, quinolines (e.g., chloroquine, quinine,phenylquinoline carboxamides), furocoumarins (e.g., psoralens andisopsoralens), ethidium, propidium, coralyne, and polycyclic aromatichydrocarbons and their oxirane derivatives;

distamycin, netropsin, other lexitropsins, Hoechst 33258 and otherHoechst dyes, DAPI (4′,6-diamidino-2-phenylindole), berenil, andtriarylmethane dyes;

aflatoxins;

spermine, spermidine, and other polyamines; and

nucleic acids or analogs which bind by sequence specific interactionssuch as triple helix formation, D-loop formation, and direct basepairing to single stranded targets. Derivatives of these compounds arealso non-limiting examples of anchor groups, where a derivative of acompound includes, but is not limited to, a compound which bears one ormore substituent of any type at any location, oxidation or reductionproducts of the compound, etc.

Examples of linkers which can be used in the invention are, but are notlimited to, compounds which include functional groups such as ester(where the carbonyl carbon of the ester is between the anchor and thesp³ oxygen of the ester; this arrangement is also called “forwardester”), “reverse ester” (where the sp³ oxygen of the ester is betweenthe anchor and the carbonyl carbon of the ester), thioester (where thecarbonyl carbon of the thioester is between the anchor and the sulfur ofthe thioester, also called “forward thioester”), reverse thioester(where the sulfur of the thioester is between the anchor and thecarbonyl carbon of the thioester, also called “reverse thioester”),forward and reverse thionoester, forward and reverse dithioic acid,sulfate, forward and reverse sulfonates, phosphate, and forward andreverse phosphonate groups. “Thioester” designates the —C(═O)—S— group;“thionoester” designates the —C(═S)—O— group, and “dithioic acid”designates the —C(═S)—S— group. The frangible linker also may include anamide, where the carbonyl carbon of the amide is between the anchor andthe nitrogen of the amide (also called a “forward amide”), or where thenitrogen of the amide is between the anchor and the carbonyl carbon ofthe amide (also called a “reverse amide”). For groups which can bedesignated as “forward” and “reverse”, the forward orientation is thatorientation of the functional groups wherein, after hydrolysis of thefunctional group, the resulting acidic function is covalently linked tothe anchor moiety and the resulting alcohol or thiol function iscovalently linked to the effector moiety. The reverse orientation isthat orientation of the functional groups wherein, after hydrolysis ofthe functional group, the resulting acidic function is covalently linkedto the effector moiety and the resulting alcohol or thiol function iscovalently linked to the anchor moiety.

The frangible linker, such as an amide moiety, also may be capable ofdegrading under conditions of enzymatic degradation, by endogenousenzymes in the biological material being treated, or by enzymes added tothe material.

Examples of effectors which can be used in the invention are, but arenot limited to, mustard groups, mustard group equivalents, epoxides,aldehydes, formaldehyde synthons, and other alkylating and cross-linkingagents. Mustard groups are defined as including mono or bishaloethylamine groups, and mono haloethylsulfide groups. Mustard groupequivalents are defined by groups that react by a mechanism similar tothe mustards (that is, by forming an aziridinium intermediate, or byhaving or by forming an aziridine ring, which can react with anucleophile), such as mono or bis mesylethylamine groups, monomesylethylsulfide groups, mono or bis tosylethylamine groups, and monotosylethylsulfide groups. Formaldehyde synthons are defined as anycompound that breaks down to formaldehyde in aqueous solution, includinghydroxymethylamines such as hydroxymethylglycine. Examples offormaldehyde synthons are given in U.S. Pat. No. 4,337,269 and inInternational Patent Application WO 97/02028. While the invention is notlimited to any specific mechanism, the effector groups, which are, orare capable of forming an electrophilic group, such as a mustard group,are believed to react with and form a covalent bond to nucleic acid.

Three embodiments of the compounds of this invention are described bythe following general formulas I, II, and III.

General formula I is:

(I)

wherein at least one of R₁, R₂, R₃, R₄, R₅, R, R₇, R₈, and R₉ is—V—W—X—E as defined below, and the remainder of R₁, R₂, R₃, R₄, R₅, R₆,R₇, R₈ and R₉ are independently selected from the group consisting of—H, —R₁₀, —O—R₁₀, —NO₂, —NH₂, —NH—R₁₀, —N(NR₁₀)₂, —F, —Cl, —Br, —I,—C(═O)—R₁₀, —C(═O)—O—R₁₀, and —O—C(═O)—R₁₀,

where —R₁₀, is independently H, —C₁₋₈alkyl, —C₁₋₈heteroalkyl, -aryl,-heteroaryl, —C₁₋₃alkyl-aryl, —C₁₋₃heteroalkyl-aryl,—C₁₋₃alkyl-heteroaryl, —C₁₋₃heteroalkyl-heteroaryl, -aryl-C₁₋₃alkyl,-aryl-C₁₋₃heteroalkyl, -heteroaryl-C₁₋₃alkyl,-heteroaryl-C₁₋₃heteroalkyl, —C₁₋₃alkyl-aryl-C₁₋₃alkyl,—C₁₋₃heteroalkyl-aryl-C₁₋₃alkyl, —C₁₋₃alkyl-heteroaryl-C₁₋₃alkyl,—C₁₋₃alkyl-aryl-C₁₋₃heteroalkyl, —C₁₋₃heteroalkyl-heteroaryl-C₁₋₃alkyl,—C₁₋₃heteroalkyl-aryl-C₁₋₃heteroalkyl,—C₁₋₃alkyl-heteroaryl-C₁₋₃heteroalkyl, or—C₁₋₃heteroalkyl-heteroaryl-C₁₋₃heteroalkyl;

V is independently —R₁₁—, —NH—R₁₁— or —N(CH₃)—R₁₁—, where —R₁₁— isindependently —C₁₋₈alkyl-, —C₁₋₈heteroalkyl-, -aryl-, -heteroaryl-,—C₁₋₃alkyl-aryl-, —C₁₋₃heteroalkyl-aryl-, —C₁₋₃alkyl-heteroaryl-,—C₁₋₃heteroalkyl-heteroaryl-, -aryl-C₁₋₃alkyl-, -aryl-C₁₋₃heteroalkyl-,-heteroaryl-C₁₋₃alkyl-, -heteroaryl-C₁₋₃heteroalkyl-,—C₁₋₃alkyl-aryl-C₁₋₃alkyl-, —C₁₋₃heteroalkyl-aryl-C₁₋₃alkyl-,—C₁₋₃alkyl-heteroaryl-C₁₋₃alkyl-, —C₁₋₃alkyl-aryl-C₁₋₃heteroalkyl-,—C₁₋₃heteroalkyl-heteroaryl-C₁₋₃alkyl-,—C₁₋₃heteroalkyl-aryl-C₁₋₃heteroalkyl-,—C₁₋₃alkyl-heteroaryl-C₁₋₃heteroalkyl-, or—C₁₋₃heteroalkyl-heteroaryl-C₁₋₃heteroalkyl-;

W is independently —C(═O)—O—, —O—C(═O)—, —C(═S)—O—, —O—C(═S)—,—C(═S)—S—, —S—C(═S)—, —C(═O)—S—, —S—C(═O)—, —O—S(═O)₂—O—, —S(═O)₂—O—,—O—S(═O)₂—, —C(═O)—NR₁₀—, —NR₁₀—C(═O)—, —O—P(═O)(—OR₁₀)—O—,—P(═O)(—OR₁₀)—O—, —O—P(═O)(—OR₁₀)—,

X is independently —R₁₁—; and

E is independently selected from the group consisting of —N(R₁₂)₂,—N(R₁₂)(R₁₃), —S—R₁₂, and

 where —R₁₂ is —CH₂CH₂—G, where each G is independently —Cl, —Br, —I,—O—S(═O)₂—CH₃, —O—S(═O)₂—CH₂—C₆H₅, or —O—S(═O)₂—C₆H₄—CH₃;

and where R₁₃ is independently-C₁₋₈alkyl, —C₁₋₈heteroalkyl, -aryl,-heteroaryl, —C₁₋₃alkyl-aryl, —C₁₋₃heteroalkyl-aryl,—C₁₋₃alkyl-heteroaryl, —C₁₋₃heteroalkyl-heteroaryl,-aryl-C₁₋₃alkyl,-aryl-C₁₋₃heteroalkyl, -heteroaryl-C₁₋₃alkyl,-heteroaryl-C₁₋₃heteroalkyl, —C₁₋₃alkyl-aryl-C₁₋₃alkyl,—C₁₋₃heteroalkyl-aryl-C₁₋₃alkyl, —C₁₋₃alkyl-heteroaryl-C₁₋₃alkyl,—C₁₋₃alkyl-aryl-C₁₋₃heteroalkyl, —C₁₋₃heteroalkyl-heteroaryl-C₁₋₃alkyl,—C₁₋₃heteroalkyl-aryl-C₁₋₃heteroalkyl,—C₁₋₃alkyl-heteroaryl-C₁₋₃heteroalkyl, or—C₁₋₃heteroalkyl-heteroaryl-C₁₋₃heteroalkyl;

and all salts and stereoisomers (including enantiomers anddiastereomers) thereof.

General formula II is:

where R₁, R₂, R₃, R₄, R₅, R₆, R₇, and R₈ are independently selected fromthe group consisting of —H, —R₁₀, —O—R₁₀, —NO₂, —NH₂, —NH—R₁₀, —N(R₁₀)₂,—F, —Cl, —Br, —I, —C(═O)—R₁₀, —C(═O)—O—R₁₀, and —O—C(═O)—R₁₀,

where —R₁₀ is independently H, —C₁₋₈alkyl, —C₁₋₈heteroalkyl, -aryl,-heteroaryl, —C₁₋₃alkyl-aryl, —C₁₋₃heteroalkyl-aryl,—C₁₋₃alkyl-heteroaryl, —C₁₋₃heteroalkyl-heteroaryl, -aryl-C₁₋₃alkyl,-aryl-C₁₋₃heteroalkyl, -heteroaryl-C₁₋₃alkyl,-heteroaryl-C₁₋₃heteroalkyl, —C₁₋₃alkyl-aryl-C₁₋₃alkyl,—C₁₋₃heteroalkyl-aryl-C₁₋₃alkyl, —C₁₋₃alkyl-heteroaryl-C₁₋₃alkyl,—C₁₋₃alkyl-aryl-C₁₋₃heteroalkyl, —C₁₋₃heteroalkyl-heteroaryl-C₁₋₃alkyl,—C₁₋₃heteroalkyl-aryl-C₁₋₃heteroalkyl,—C₁₋₃alkyl-heteroaryl-C₁₋₃heteroalkyl, or—C₁₋₃heteroalkyl-heteroaryl-C₁₋₃heteroalkyl;

R₂₀ is —H or —CH₃; and

R₂₁ is —R₁₁—W—X—E,

where —R₁₁— is independently —C₁₋₈alkyl-, —C₁₋₈heteroalkyl-, -aryl-,-heteroaryl-, —C₁₋₃alkyl-aryl-, —C₁₋₃heteroalkyl-aryl-,—C₁₋₃alkyl-heteroaryl-, —C₁₋₃heteroalkyl-heteroaryl-, -aryl-C₁₋₃alkyl-,-aryl-C₁₋₃heteroalkyl-, -heteroaryl-C₁₋₃alkyl-,-heteroaryl-C₁₋₃heteroalkyl-, —C₁₋₃alkyl-aryl-C₁₋₃alkyl-,—C₁₋₃heteroalkyl-aryl-C₁₋₃alkyl-, —C₁₋₃alkyl-heteroaryl-C₁₋₃alkyl-,—C₁₋₃alkyl-aryl-C₁₋₃heteroalkyl-,—C₁₋₃heteroalkyl-heteroaryl-C₁₋₃alkyl-,—C₁₋₃heteroalkyl-aryl-C₁₋₃heteroalkyl-,—C₁₋₃alkyl-heteroaryl-C₁₋₃heteroalkyl-, or—C₁₋₃heteroalkyl-heteroaryl-C₁₋₃heteroalkyl-;

W is independently —C(═O)—O—, —O—C(═O)—, —C(═S)—O—, —O—C(═S)—,—C(═S)—S—, —S—C(═S)—, —C(═O)—S—, —S—C(═O)—, —O—S(═O)₂—O—, —S(═O)₂—O—,—O—S(═O)₂—, —C(═O)—NR₁₀—, —NR₁₀—C(═O)—, —O—P(═O)(—OR₁₀)—O—,—P(═O)(—OR₁₀)—O—, —O—P(═O)(—OR₁₀)—;

X is independently —R₁₁—; and

E is independently selected from the group consisting of —N(R₁₂)₂,—N(R₁₂)(R₁₃), —S—R₁₂, and

 where —R₁₂ is —CH₂CH₂—G, where each G is independently —Cl, —Br, —I,—O—S(═O)₂—CH₃, —O—S(═O)₂—CH₂—C₆H₅, or —O—S(═O)₂—C₆H₄—CH₃;

and where R₁₃ is independently —C₁₋₈alkyl, —C₁₋₈heteroalkyl, -aryl,-heteroaryl, —C₁₋₃alkyl-aryl, —C₁₋₃heteroalkyl-aryl,—C₁₋₃alkyl-heteroaryl, —C₁₋₃heteroalkyl-heteroaryl, -aryl-C₁₋₃alkyl,-aryl-C₁₋₃heteroalkyl, -heteroaryl-C₁₋₃alkyl,-heteroaryl-C₁₋₃heteroalkyl, —C₁₋₃alkyl-aryl-C₁₋₃alkyl,—C₁₋₃heteroalkyl-aryl-C₁₋₃alkyl, —C₁₋₃alkyl-heteroaryl-C₁₋₃alkyl,—C₁₋₃alkyl-aryl-C₁₋₃heteroalkyl, —C₁₋₃heteroalkyl-heteroaryl-C₁₋₃alkyl,—C₁₋₃heteroalkyl-aryl-C₁₋₃heteroalkyl,—C₁₋₃alkyl-heteroaryl-C₁₋₃heteroalkyl, or—C₁₋₃heteroalkyl-heteroaryl-C₁₋₃heteroalkyl;

and all salts and stereoisomers (including enantiomers anddiastereomers) thereof.

General formula III is:

wherein at least one of R₄₄, R₅₅, R₃, R₄, R₅, and R₈ is —V—W—X—E, andthe remainder of R₄₄, R₅₅, R₃, R₄, R₅, and R₈ are independently selectedfrom the group consisting of —H, —R₁₀, —O—R₁₀, —NO₂, —NH₂, —NH—R₁₀,—N(R₁₀)₂, —F, —Cl, —Br, —I, —C(═O)—R₁₀, —C(═O)—O—R₁₀, and —O—C(═O)—R₁₀,

where —R₁₀ is independently H, —C₁₋₈alkyl, —C₁₋₈heteroalkyl, -aryl,-heteroaryl, —C₁₋₃alkyl-aryl, —C₁₋₃heteroalkyl-aryl,—C₁₋₃alkyl-heteroaryl, —C₁₋₃heteroalkyl-heteroaryl, -aryl-C₁₋₃alkyl,-aryl-C₁₋₃heteroalkyl, -heteroaryl-C₁₋₃alkyl,-heteroaryl-C₁₋₃heteroalkyl, —C₁₋₃alkyl-aryl-C₁₋₃alkyl,—C₁₋₃heteroalkyl-aryl-C₁₋₃alkyl, —C₁₋₃alkyl-heteroaryl-C₁₋₃alkyl,—C₁₋₃alkyl-aryl-C₁₋₃heteroalkyl, —C₁₋₃heteroalkyl-heteroaryl-C₁₋₃alkyl,—C₁₋₃heteroalkyl-aryl-C₁₋₃heteroalkyl,—C₁₋₃alkyl-heteroaryl-C₁₋₃heteroalkyl, or—C₁₋₃heteroalkyl-heteroaryl-C₁₋₃heteroalkyl;

V is independently —R₁₁—, —NH—R₁₁— or —N(CH₃)—R₁₁—, where —R₁₁— isindependently —C₁₋₈alkyl-, —C₁₋₈heteroalkyl-, -aryl-, -heteroaryl-,—C₁₋₃alkyl-aryl-, —C₁₋₃heteroalkyl-aryl-, —C₁₋₃alkyl-heteroaryl-,—C₁₋₃heteroalkyl-heteroaryl-, -aryl-C₁₋₃alkyl-, -aryl-C₁₋₃heteroalkyl-,-heteroaryl-C₁₋₃alkyl-, -heteroaryl-C₁₋₃heteroalkyl-,—C₁₋₃alkyl-aryl-C₁₋₃alkyl-, —C₁₋₃heteroalkyl-aryl-C₁₋₃alkyl-,—C₁₋₃alkyl-heteroaryl-C₁₋₃alkyl-, —C₁₋₃alkyl-aryl-C₁₋₃heteroalkyl-,—C₁₋₃heteroalkyl-heteroaryl-C₁₋₃alkyl-,—C₁₋₃heteroalkyl-aryl-C₁₋₃heteroalkyl-,—C₁₋₃alkyl-heteroaryl-C₁₋₃heteroalkyl-, or—C₁₋₃heteroalkyl-heteroaryl-C₁₋₃heteroalkyl-;

W is independently —C(═O)—O—, —O—C(═O)—, —C(═S)—O—, —O—C(═S)—,—C(═S)—S—, —S—C(═S)—, —C(═O)—S—, —S—C(═O)—, —O—S(═O)₂—O—, —S(═O)₂—O—,—O—S(═O)₂—, —C(═O)—NR₁₀—, —NR₁₀—C(═O)—, —O—P(═O)(—OR₁₀)—O—,—P(═O)(—OR₁₀)—O—, —O—P(═O)(—OR₁₀)—;

X is independently —R₁₁—; and

E is independently selected from the group consisting of —N(R₁₂)₂,—N(R₁₂)(R₁₃), —S—R₁₂, and

 where —R₁₂ is —CH₂CH₂—G, where each G is independently —Cl, —Br, —I,—O—S(═O)₂—CH₃, —O—S(═O)₂—CH₂—C₆H₅, or —O—S(═O)₂—C₆H₄—CH₃;

and where R₁₃ is independently —C₁₋₈alkyl —C₁₋₈heteroalkyl, -aryl,-heteroaryl, —C₁₋₃alkyl-aryl, —C₁₋₃heteroalkyl-aryl,—C₁₋₃alkyl-heteroaryl, —C₁₋₃heteroalkyl-heteroaryl, -aryl-C₁₋₃alkyl,-aryl-C₁₋₃heteroalkyl, -heteroaryl-C₁₋₃alkyl,-heteroaryl-C₁₋₃heteroalkyl, —C₁₋₃alkyl-aryl-C₁₋₃alkyl,—C₁₋₃heteroalkyl-aryl-C₁₋₃alkyl, —C₁₋₃alkyl-heteroaryl-C₁₋₃alkyl,—C₁₋₃alkyl-aryl-C₁₋₃heteroalkyl, —C₁₋₃heteroalkyl-heteroaryl-C₁₋₃alkyl,—C₁₋₃heteroalkyl-aryl-C₁₋₃heteroalkyl,—C₁₋₃alkyl-heteroaryl-C₁₋₃heteroalkyl, or—C₁₋₃heteroalkyl-heteroaryl-C₁₋₃heteroalkyl;

and all salts and stereoisomers (including enantiomers anddiastereomers) thereof.

It will be appreciated that, in general formula I above, the acridinenucleus is the anchor moiety, the —V—W—X— group(s) comprises thefrangible linker, and the E group(s) is the effector group. Similarly,in general formula III above, the psoralen nucleus is the anchor moiety,the —V—W—X— group(s) comprises the frangible linker, and the E group(s)is the effector group. General formula II is a subset of general formulaI.

An exemplary compound of the invention is the structure below,designated IV:

In IV, a 2-carbomethoxyacridine ring system serves as the anchor moietyvia intercalation. A bis (chloroethyl) amine group serves as theeffector moiety, which can alkylate nucleic acid; the nitrogen mustardhydrolyzes if it does not react with nucleic acid. The linker is—NH—CH₂CH₂—C(═O)—O—CH₂CH₂—. In aqueous solution at physiological pH,this ester-containing linker hydrolyzes within hours. Changing the pH ofthe solution changes the rate at which the linker hydrolyzes; for thecorresponding alcohol analog of IV (where the —Cl atoms of IV arereplaced with —OH groups), ≦1% hydrolysis of the ester linkage isobserved at pH 3 after 100 minutes at 37° C.; at pH 8, more than 50%hydrolysis of the ester linkage is observed after 100 minutes at 37° C.The resulting hydrolysis products of IV areN-(2-carbomethoxy-9-acridinyl)-β-alanine and triethanolamine:

where the 2-carbomethoxyacridine bears β-alanine as a linker fragment,and the effector breakdown product bears an ethanol group as a linkerfragment.

At physiological pH values, the carboxylate of the β-alanine will benegatively charged, a feature which decreases the tendency of theattached 2-carbomethoxyacridine group to intercalate into a negativelycharged nucleic acid molecule. This lowers the mutagenicity ofN-(2-carbomethoxy-9-acridinyl)-β-alanine relative to 9-aminoacridine.This potential for lowering the mutagenicity of the anchor fragmentillustrates one advantage provided by the frangible linker.

Another advantage of the frangible linker in compounds similar to IV isthat the hydrolysis rate can be adjusted by varying the length of thelinker arm between the 9-aminoacridine anchor moiety and the esterfunction. As described in Example 7 and Tables III and IV below, anincrease in the number of methylene groups between the aminoacridineanchor and the ester group results in a decrease in the amount ofhydrolysis seen in aqueous solution, at pH 8, 37° C., for diol analogsof certain compounds of the invention (where the —Cl atoms of themustards are replaced with —OH groups).

Examples of the compounds of the invention are given below, asillustration and not as any limitation on the invention.

Applications

Examples of uses of the compounds of the invention include, but are notlimited to: addition of the compounds of the invention in solid orsolution form to biological materials, to inactivate pathogens presentin the biological materials; immersion or other treatment of a materialfor biological use in a solution of the compounds of the invention, toinactivate pathogens present in or on the material; and inclusion ofcompounds of the invention in targeted liposomes, to direct thecompounds to particular cells in order to damage the nucleic acid ofthose cells.

It should be noted that while the compounds of the invention aredesigned to hydrolyze under certain conditions, they are stable underother conditions. It is desirable for the frangible linker and theeffector group(s) to be relatively stable under certain conditions usedfor storage. Examples of manners in which the compounds may be storedinclude, but are not limited to, dry solids, oils with low watercontent, frozen aqueous solutions, frozen non-aqueous solutions,suspensions, and solutions which do not permit hydrolysis of thefrangible linker or the effector group(s), for example liquidnon-aqueous solutions. The compounds may be stored at temperatures at orbelow 0° C. (e.g., in a freezer), or at temperatures above 0° C. (e.g.,in a refrigerator or at ambient temperatures). The compounds preferablyare stable under the storage conditions for a period of between threedays and one year, between one week and one year, between one month andone year, between three months and one year, between six months and oneyear, between one week and six months, between one month and six months,between three months and six months, between one week and three months,or between one month and three months. The stability of the compoundswill be determined both by the temperature at which they are stored, andby the state in which they are stored (e.g., non-aqueous solution, drysolid).

Conditions for Pathogen Inactivation

Conditions for treating biological materials with a pathogeninactivating compound may be selected based on the selected material andthe inactivating compound. Typical concentrations of pathogeninactivating compound for the treatment of biological materials such asblood products are on the order of about 0.1 μM to 5 mM, for exampleabout 500 μM. For example, a concentration of pathogen inactivatingcompound may be used which is sufficient to inactivate at least about 1log, or at least about 2 logs, or for example, at least about 3 to 6logs of a pathogen in the sample. In one embodiment, the pathogeninactivating compound produces at least 1 log kill at a concentration ofno greater than about 500 μM, more preferably at least 3 logs kill at nogreater than 500 μM concentration. In another non-limiting example, thepathogen inactivating compound may have at least 1 log kill, andpreferably at least 6 logs kill at a concentration of about 0.1 μM toabout 3 mM.

Incubation of blood products with the pathogen inactivating compound canbe conducted for example, for about 5 minutes to 72 hours or more, orabout 1 to 48 hours, for example, about 1 to 24 hours, or, for example,about 8 to 20 hours. For red blood cells, the incubation is typicallyconducted at a temperature of about 2° C. to 37° C., preferably about18° C. to 25° C. For platelets, the temperature is preferably about 20to 24° C. For plasma, the temperature may be about 0 to 60° C.,typically about 0-24° C. The pH of the material being treated ispreferably about 4 to 10, more preferably about 6 to 8.

One embodiment of the invention encompasses compounds and methods foruse in inactivating pathogens in blood or blood products, and apreferred set of storage conditions for this purpose would be thoseconditions that allow the convenient storage and use of the compounds atblood banks.

Under the conditions used for pathogen inactivation in or on a material,the frangible linker and effector group(s) will undergo hydrolysis orreaction. The hydrolysis, of both the frangible linker and the effectorgroups(s), preferably is slow enough to enable the desired amount ofpathogen inactivation to take place. The time required for pathogeninactivation may be, for example, about 5 minutes to 72 hours.

Treatment of Red Blood Cells

Preferably, treatment of red blood cell containing materials with thepathogen inactivating compound does not damage red blood cell functionor modify red blood cells after treatment. The lack of a substantiallydamaging effect on red blood cell function may be measured by methodsknown in the art for testing red blood cell function. For example, thelevels of indicators such as intracellular ATP (adenosine5′-triphosphate), intracellular 2,3-DPG (2,3-diphosphoglycerol) orextracellular potassium may be measured, and compared to an untreatedcontrol. Additionally hemolysis, pH, hematocrit, hemoglobin, osmoticfragility, glucose consumption and lactate production may be measured.

Methods for determining ATP, 2,3-DPG, glucose, hemoglobin, hemolysis,and potassium are available in the art. See for example, Davey et al.,Transfusion, 32:525-528 (1992), the disclosure of which is incorporatedherein. Methods for determining red blood cell function are alsodescribed in Greenwalt et al., Vox Sang, 58:94-99 (1990); Hogman et al.,Vox Sang, 65:271-278 (1993); and Beutler et al., Blood, Vol. 59 (1982)the disclosures of which are incorporated herein by reference.Extracellular potassium levels may be measured using a Ciba Coming Model614 K⁺/Na⁺ Analyzer (Ciba Coming Diagnostics Corp., Medford, Mass.). ThepH can be measured using a Ciba Coming Model 238 Blood Gas Analyzer(Ciba Coming Diagnostics Corp., Medford, Mass.).

Binding of species such as IgG, albumin, and IgM to red blood cells alsomay be measured using methods available in the art. Binding of moleculesto red blood cells can be detected using antibodies, for example toacridine and IgG. Antibodies for use in assays can be obtainedcommercially, or can be made using methods available in the art, forexample as described in Harlow and Lane, “Antibodies, a LaboratoryManual, Cold Spring Harbor Laboratory,” 1988, the disclosure of which isincorporated herein. For example, anti-IgG is commercially availablefrom Caltag, Burlingame, Calif.; Sigma Chemical Co., St. Louis, Mo. andLampire Biological Laboratory, Pipersvelle, Pa.

In a method of treatment of a material comprising red blood cells withthe pathogen inactivating compound, preferably the level ofextracellular potassium is not greater than 3 times, more preferably nomore than 2 times the amount exhibited in the untreated control after 1day. In another embodiment, preferably, hemolysis of the treated redblood cells is less than 3% after 28 day storage, more preferably lessthan 2% after 42 day storage, and most preferably less than or equal toabout 1% after 42 day storage at 4° C.

Biological Materials

A variety of biological materials may be treated with a pathogeninactivating compound. Biological materials include blood products suchas whole blood, packed red blood cells, platelets and fresh or frozenplasma. Blood products further encompass plasma protein portion,antihemophilic factor (Factor VIII), Factor IX and Factor IX complex,fibrinogens, Factor XIII, prothrombin and thrombin, immunoglobulins(such as IgG, IgA, IgD, IgE and IgM and fragments thereof), albumin,interferon, and lymphokines. Also contemplated are synthetic bloodproducts.

Other biological materials include vaccines, recombinant DNA producedproteins and oligopeptide ligands. Also encompassed are clinical samplessuch as urine, sweat, sputum, feces, spinal fluid. Further encompassedare synthetic blood or blood product storage media.

Reducing the Concentration of Compounds in Materials after Treatment

The concentration of the pathogen inactivating compound in a biologicalmaterial, such as a blood product, can be reduced after the treatment.Methods and devices which may be used are described in PCT/US96/09846;U.S. Ser. No. 08/779,830, filed Jan. 6, 1997; and in the co-filedapplication, “Methods and Devices for the Reduction of Small OrganicCompounds from Blood Products”, PCT/US98/00531, filed Jan. 6, 1998, thedisclosures of each of which are incorporated herein by reference intheir entirety.

Quenching

In another embodiment the compounds of the invention may be used incombination with a quencher. Methods for quenching undesired sidereactions of pathogen inactivating compounds in biological materials aredescribed in the cofiled U.S. Provisional Application Serial No.60/070,597, filed Jan. 6, 1998, Attorney Docket No. 282173000600,“Methods for Quenching Pathogen Inactivators in Biological Materials,”the disclosure of which is incorporated herein. Disclosed in the cofiledapplication are methods for quenching undesired side reactions of apathogen inactivating compound that includes a functional group whichis, or which is capable of forming, an electrophilic group. In thisembodiment, the material is treated with the pathogen inactivatingcompound and a quencher, wherein the quencher comprises a nucleophilicfunctional group that is capable of covalently reacting with theelectrophilic group. Preferred quenchers are thiols, such asglutathione.

EXAMPLES

The following specific examples are presented to illustrate thepreparative methods for representative compounds useful in the method ofthis invention, to provide relevant data regarding the compounds usefulto the practitioner, and to illustrate the manner in which theeffectivity of the compounds is determined, and are not to be construedas limiting the scope of the invention. All NMR spectra were recorded ona Varian 200 MHz instrument in CDCl₃ unless otherwise noted; chemicalshifts are reported versus tetramethylsilane (TMS). IR spectra wererecorded with a Perkin Elmer FTIR. HPLC was carried out with a YMC C8column in a gradient mode using 5 mM aq. H₃PO₄ as mobile phase A and 5mM CH₃CN as mobile phase B. Samples were prepared in DMSO or ethanol andkept at ≧15° C. prior to injection.

Table II indicates the designation of compound number used for thevarious compounds.

TABLE II COMPOUND NUMBER CMEMICAL NAME IV β-alanine,N-(2-carbomethoxyacridin-9-yl),2-[bis(2- chloroethyl)amino]ethyl ester Vβ-alanine, N-(acridin-9-yl), 2-[bis(2-chloroethyl)amino] ethyl ester VI4-aminobutyric acid N-[(2-carbomethoxyacridin-9-yl), 2-[bis(2-chloroethyl)amino]ethyl ester VII 5-aminovaleric acidN-[(2-carbomethoxyacridin-9-yl), 2- [bis(2-chloroethyl)amino]ethyl esterVIII β-alanine, N-(2-carbomethoxyacridin-9-yl), 3-[bis(2-chloroethyl)amino]propyl ester IX β-alanine, N-(4-methoxyacridin-9-yl),2-[bis(2- chloroethyl)amino]ethyl ester X β-alanine,N-(3-chloro-4-methylacridin-9-yl), 2-[bis(2- chloroethyl)amino]ethylester XI β-alanine, [N,N-bis(2-chloroethyl)], 3-[(6-chloro-2-methoxyacridin-9-yl)amino]propyl ester XII β-alanine,[N,N-bis(2-chloroethyl)], 2-[(6-chloro-2-methoxyacridin-9-yl)amino]ethyl ester XIII β-alanine,N-(6-chloro-2-methoxyacridin-9-yl), 2-[bis(2- chloroethyl)amino]ethylester XIV [N,N-bis(2-chloroethyl)]-2-aminoethyl 4,5′,8-trimethyl-4′-psoralenacetate XV β-alanine, N-(acridin-9-yl),2-[bis(2-chloroethyl)amino] ethyl amide

Example 1 Synthesis of β-Alanine, N-(2-Carbomethoxyacridin-9-yl),2-[bis(2-Chloroethyl)amino]ethyl Ester Dihydrochloride (Compound IV,)

Step A. β-Alanine, N-(tert-Butoxycarbonyl),2-[bis(2-Hydroxyethyl)amino]ethyl Ester

To a stirred solution of N-(tert-butoxycarbonyl)-β-alanine (20.3 g, 107mmol) and 4-methylmorpholine (13.0 mL, 12.0 g, 119 mmol) in dry THF (200mL) at −15° C. under N₂ was added isobutyl chloroformate (13.9 mL, 14.6g, 107 mmol) resulting in the immediate formation of a white precipitate(4-methylmorpholine·HCl). The reaction mixture was stirred at −15° C.for 5 min. followed by the transfer of the reaction mixture to a flaskcontaining a stirred solution of triethanolamine (48.3 g, 324 mmol) indry THF (150 mL) at −15° C. The reaction mixture was allowed to warm to23° C. and stirred for an additional 1.5 h. followed by removal of theprecipitate by vacuum filtration. The THF was then removed in vacuo fromthe filtrate and the remaining viscous yellow oil was partitionedbetween water (500 mL) and EtOAc (5×150 mL). The combined organic layerswere dried over Na₂SO₄. Removal of solvent in vacuo gave 25.8 g (75% )of the desired product, β-alanine, N-(tert-butoxycarbonyl),2-[bis(2-hydroxyethyl)amino]ethyl ester, as a pale yellow oil. ¹H NMR: δ5.32 (br s, 1H), 4.18 (t, J=5.4 Hz, 2H), 3.58 (t, J=5.1 Hz, 4H),3.37-3.23 (m, 2H), 2.80 (t, J=5.4 Hz, 2H), 2.69 (t, J=5.1 Hz, 4H), 2.51(t, J=6.0 Hz, 2H), 1.41 (s, 9H). The hydroxyl protons were not observed.¹³C NMR: δ 173.0, 156.4, 79.8, 63.3, 60.2, 57.3, 54.1, 36.7, 35.3, 28.8.

Step B. β-Alanine, N-(tert-Butoxycarbonyl),2-[bis(2-tert-Butyldimethylsilyloxyethyl)amino]ethyl Ester

A stirred solution of the β-alanine, N-(tert-butoxycarbonyl),2-[bis(2-hydroxyethyl)amino]ethyl ester from step A (22.7 g, 70.9 mmol)and imidazole (11.1 g, 163 mmol) in acetonitrile (70 mL) under N₂ wascooled to 0° C. Tert-butyldimethylsilyl chloride (534 mg, 3.54 mmol) wasthen added and the reaction mixture was stirred for an additional 5 min.at 0° C. The reaction mixture was allowed to warm to 23° C. and stirredfor 2 h followed by removal of the resultant white precipitate(imidazole·HCl) by vacuum filtration. The acetonitrile was removed invacuo from the filtrate and the remaining material was partitionedbetween saturated brine (600 mL) and EtOAc (3×200 mL). The combinedorganic layers were dried over Na₂SO₄. Removal of solvent in vacuo gave35.2 g (90% ) of the desired product, β-alanine,N-(tert-butoxycarbonyl),2-[bis(2-tertbutyldimethylsilyloxyethyl)amino]ethyl ester, as a yellowoil. ¹H NMR: δ 5.29 (br s, 1H), 4.14 (t, J=6.0 Hz, 2H), 3.65 (t, J=6.3Hz, 4H), 3.37 (apparent q, 2H), 2.85 (t, J=6.0 Hz, 2H), 2.71 (t, J=6.3Hz, 4H), 2.49 (t, J=5.9 Hz, 2H), 1.42 (s, 9H), 0.88 (s, 18H), 0.03 (s,12H); ¹³C NMR: δ 172.7, 156.3, 79.7, 63.3, 62.4, 57.7, 54.3, 36.7, 35.3,28.9, 26.4, 18.7, −4.9.

Step C. β-Alanine, 2-[bis(2-tert-Butyldimethylsilyloxyethyl)amino]ethylEster

To a flask containing β-alanine, N-(tert-butoxycarbonyl),2-[bis(2-tertbutyldimethylsilyloxyethyl)amino]ethyl ester from step B(3.01 g, 5.48 mmol) was added neat trifluoroacetic acid (5 mL) resultingin the evolution of CO₂ gas. The reaction mixture was stirred for 5 min.and the trifluoroacetic acid was removed in vacuo. The remainingmaterial was partitioned between saturated NaHCO₃ (100 mL) and EtOAc(3×30 mL). The combined organic layers were dried over Na₂SO₄. Removalof solvent in vacuo gave 2.45 g (100% ) of the desired product,β-alanine, 2-[bis(2-tert-butyldimethylsilyloxyethyl)amino]ethyl ester,as a pale yellow oil. ¹H NMR: δ 4.12 (t, J=6.0 Hz, 2H), 3.63 (t, J=6.4Hz, 4H), 2.96 (t, J=6.2 Hz, 2H), 2.84 (t, J=6.0 Hz, 2H), 2.69 (t, J=6.4Hz, 4H), 2.44 (t, J=6.2 Hz, 2H), 0.86 (s, 18H), 0.03 (s, 12H). The amineprotons were not observed. ¹³C NMR (CDCl₃): δ 173.0, 63.4, 62.6, 57.9,54.4, 38.4, 38.1, 26.4, 18.7, −4.9.

Step D. β-Alanine, N-(2-Carbomethoxyacridin-9-yl),2-[bis(2-Hydroxyethyl)amino]ethyl Ester

The β-alanine, 2-[bis(2-tert-butyldimethylsilyloxyethyl)amino]ethylester (736 mg, 1.64 mmol) was reacted with methyl9-methoxyacridine-2-carboxylate (669 mg, 2.50 mmol) by stirring in 10 mLof CHCl₃ for 12.5 h at room temperature. The precipitate (acridone) wasthen filtered off and the filtrate partitioned between saturated aqueousNaHCO₃ (100 mL) and CHCl₃ (3×35 mL). The combined organic layers weredried over Na₂SO₄ and concentrated in vacuo to give 1.61 g of viscousbrown oil. Deprotection of the resultant diol was carried out bydissolving the crude intermediate in 3.0 mL of THF under N₂ and, uponcooling to 0° C., treating with HF/pyridine (1.0 mL). The solution wasallowed to warm to room temperature with stirring for 1 h. The volatileswere removed in vacuo and the residue was partitioned between saturatedaqueous NaHCO₃ (100 mL) and CHCl₃ (3×35 mL). The combined organic layerswere dried and concentrated to give 649 mg of a brownish yellow solid.Preparative TLC (C-18, CH₃CN) gave a 20% yield of the desired diol,β-alanine, N-(2-carbomethoxyacridin-9-yl),2-[bis(2-hydroxyethyl)amino]ethyl ester (>80% pure by HPLC); ¹H NMR: δ8.82 (s, 1H), 8.21-7.94 (m, 2H), 7.94-7.72 (m, 2H), 7.59 (apparent t,1H), 7.23 (apparent t, 1H), 4.30-4.18 (m, 2H), 4.18-4.05 (m, 2H), 3.89(s, 3H), 3.69-3.50 (m, 4H), 2.92-2.73 (m, 4H), 2.73-2.55 (m, 4H). Theamine and hydroxyl protons were not observed.

Step E. β-Alanine, N-(2-Carbomethoxyacridin-9-yl),2-[bis(2-Chloroethyl)amino]ethyl Ester Dihydrochloride

Conversion of β-alanine, N-(2-carbomethoxyacridin-9-yl),2-[bis(2-hydroxyethyl)amino]ethyl ester to the dichloro compound wasachieved by a method similar to that of Peck, et al. (J. Am. Chem. Soc.1959, 81: 3984). A yellow solution of the product from step D (41 mg,0.090 mmol) in neat SOCl₂ (6 mL) was stirred at room temperature for 20hours. The SOCl₂ was then removed in vacuo to give a yellow solid(dihydrochloride salt). The material was then partitioned betweensaturated NaHCO₃ (50 mL) and CH₂Cl₂ (3×20 mL). The combined organiclayers were dried over Na₂SO₄. Removal of solvent in vacuo gave 35.4 mgof the dichloro compound free base as an orange gum. ¹H NMR: δ 8.82 (s,1H), 8.20-7.83 (m, 4H), 7.5 (apparent t, 1H), 7.25 (apparent t, 1H),4.36-4.15 (m, 4H), 3.93 (s, 3H), 3.48 (t, J=6.9 Hz, 4H), 3.06-2.77 (m,4H), 2.86 (t, J=6.9 Hz, 4H). The amine proton was not observed. ¹³CNMR:δ 172.3, 166.6, 155.2, 146.5, 144.6, 133.1, 131.6, 128.7, 124.6, 124.3,116.1, 114.3, 63.7, 57.2, 53.5, 52.9, 46.3, 42.5, 35.2. No other carbonswere observed. The HCl salt was precipitated from CH₂Cl₂ by addition of1M HCl in ether to give β-alanine, N-(2-carbomethoxyacridin-9-yl),2-[bis(2-chloroethyl)amino]ethyl ester dihydrochloride (Compound IV,) asa yellow solid (81% pure by HPLC).

β-Alanine, N-(acridin-9-yl), 2-[bis(2-chloroethyl)amino]ethyl esterdihydrochloride, (Compound V) was prepared in a similar manner. Thususing 9-methoxyacridine in place of methyl9-methoxyacridine-2-carboxylate in Step D, the intermediate diol wasobtained (7.1% ) as a yellow oil (74% pure by HPLC). ¹H NMR: δ 8.14 (d,J=7.5 Hz, 2H), 7.93 (d, J=8.6 Hz, 2H), 7.52 (apparent t, 2H), 7.23(apparent t, 2H), 4.36-4.08 (m, 4H), 3.76-3.5 (m, 4H), 3.08-2.60 (m,8H). The amine and hydroxyl protons were not observed.

A solution of the intermediate diol (37.3 mg, 0.0793 mmol) in thionylchloride (4.0 mL) was stirred at 23° C. for 7.5 h. The thionyl chloridewas removed in vacuo to give a yellow oil. The material was dissolved inethanol (4 mL) and the solvent removed in vacuo. The material was thendissolved in CH₂Cl₂ (4 mL) and solvent removed in vacuo; this step wasrepeated twice. The material was then triturated with hexane (3×4 mL) togive 40.0 mg (42% pure by HPLC) of the product in the form of a yellowhydroscopic glassy solid. Some of the material was converted to the freeamine for analytical purposes by partitioning between saturated NaHCO₃and CH₂Cl₂ followed by drying the combined organic layers over Na₂SO₄and removal of the solvent in vacuo. ¹H NMR: δ 8.21-8.00 (m, 4H), 7.66(apparent t, 2H), 7.38 (apparent t, 2H), 4.26-4.12 (m, 2H), 4.12-3.98(m, 2H), 3.43 (t, J=6.9 Hz, 4H), 2.96-2.68 (m, 8H). The amine proton wasnot observed.

Following the above procedure but replacingN-(tert-butoxycarbonyl)-β-alanine withN-(tert-butoxycarbonyl)-4-aminobutyric acid led to the preparation of4-aminobutyric acid N[(2-carbomethoxyacridin-9-yl),2-[bis(2-chloroethyl)amino]ethyl ester dihydrochloride, Compound VI (78%pure by HPLC). ¹H NMR: δ 8.89 (s, 1), 8.12 (apparent t, 2), 7.93-7.80(m, 2), 7.59 (apparent q, 1), 7.36-7.20 (m, 1), 4.16 (t, 2, J=5.7 Hz),4.07-3.92 (m, 2), 3.97 (s, 3), 3.46 (t, 4, J=6.9 Hz), 2.93-2.80 (m, 6),2.60 (t, 2, J=6.5 Hz), 2.29-2.12 (m, 2). The amine proton was notobserved.

Example 2

Substituting the triethanolamine in Example 1, Step A with3-[N,N-Bis(2-tertbutyldimethylsilyloxyethyl)]aminopropanol, and thencontinuing from step C, led to the preparation of β-alanine,N-(2-carbomethoxy-acridin-9-yl), 3-[bis(2-chloroethyl)amino]propyl esterdihydrochloride, Compound VIII, (63% pure by HPLC). ¹H NMR: δ 8.91 (s,1), 8.20-7.93 (m, 4), 7.18 (apparent t, 1), 7.39 (apparent t, 1), 4.30(m, 4), 3.96 (s, 3), 3.48 (t, 4, J=6.9 Hz), 2.88-2.60 (m, 2), 2.83 (t,4, J=6.9 Hz), 2.62 (t, 2, J=6.7 Hz), 1.85-1.68 (m, 2). The amine protonwas not observed.

Example 3

The compounds synthesized in Example 1 can also be prepared by thefollowing method:

Synthesis of β-Alanine, N-(Acridin-9-yl),2-[bis(2-Chloroethyl)amino]ethyl Ester Dihydrochloride (Compound V):Method II

Step A: β-Alanine, N-(Acridin-9-yl), Methyl Ester Hydrochloride

9-Chloroacridine (11.7 g, Organic Synthesis, Coll. Vol III, pg 57),β-alanine methyl ester hydrochloride (9.9 g) and sodium methoxide (3.26g) were combined and 60 mL of methanol was added. The mixture wasstirred with a magnetic stirrer and refluxed for 5.5 h. Heat was removedand the suspension was filtered while warm (≦35° C.). The solid saltswere rinsed with about 10 mL of additional methanol and the combineddark green filtrate was concentrated to give 21 g of a moistgreenish-yellow solid.

The solid was dissolved in 350 mL of boiling 2-propanol and allowed tocrystallize at room temperature. The resulting crystals were rinsed withabout 15 mL of 2-propanol and 15 mL of hexane, then air dried to give15.5 g of bright yellow product, β-alanine, N-(acridin-9-yl),methylester hydrochloride, (yield 78.5% ). ¹H NMR: δ 1.9 (br s, 2H);3.24 (t, J=7.0 Hz, 2H); 3.76 (s, 3H); 4.45 (br s, 2H); 7.23 (app. t, J=8Hz, 2H); 7.49 (app. t, J=8 Hz, 2H); 8.11 (d, J=8.4 Hz, 2H); 8.30 (d,J=8.4 Hz, 2H); 9.68 (br s, 0.5H). IR: 1574 (s), 1691 (s), 1726 (s), 2336(m), 2361 (m), 3227 (m).

Step B: β-Alanine, N-(Acridin-9-yl), 2-[bis(2-Hydroxyethyl)amino]ethylEster Dihydrochloride

The β-alanine, N-(acridin-9-yl), methyl ester hydrochloride, from StepA, (5.00 g) was partitioned between toluene (750 mL), saturated aqueousNa₂CO₃ (200 mL) and H₂O (50 mL). The aqueous layer was extracted againwith toluene (3×250 mL) and the organic layers were combined and washedwith saturated aqueous Na₂CO₃ (50 mL). The volume of toluene was reducedto about 100 mL by rotary evaporation. Triethanolamine (30 mL) was thenadded to form a partially immiscible system. A solution of NaOMe (50 mg)in MeOH (2 mL) was then added. Solvents were quickly removed from thereaction mixture by rotary evaporation with agitation at roomtemperature. After the solvent was removed the reaction mixture was leftunder vacuum for another 1-1.5 h to give a syrupy solution.

The crude mixture was partitioned between CH₂Cl₂ (200 mL) and brine (200mL) to remove excess triethanolamine. The brine layer was extracted withCH₂Cl₂ (5×100 mL). The organic layers were combined and washed withbrine (50 mL) then extracted with 0.5M HCl (2×100 mL). The aqueous acidlayers were combined and washed with CH₂Cl₂ (50 mL). The acid solutionwas made basic with powdered K₂CO₃(s) in the presence of CH₂Cl₂ (200mL). The organic layer was separated and the aqueous layer was extractedagain with CH₂Cl₂ (5×100 mL). The combined organic layers were washedwith brine (50 mL), dried with anhydrous Na₂SO₄(s), and stripped to givecrude diol free amine (5.02 g), a sticky yellow gum. This material wasidentical by NMR to that prepared in Example 1 by an alternateprocedure.

A portion of the above crude (0.400 g) was vigorously stirred withisopropanol (100 mL) and acidified with 1M HCl in ether. The slurry waschilled and the first precipitate was discarded. After removing half thesolvent the second set of crystals gave β-alanine, N(acridin-9-yl),2-[bis(2-hydroxyethyl)amino]ethyl ester dihydrochloride as a brightyellow crystalline solid (0.200 g) >95% pure by HPLC. ¹H NMR: δ 8.11(apparent t, 4H), 7.69 (apparent t, 2H), 7.41 (apparent t, 2H), 4.23 (t,J=5.4 Hz, 2H), 4.03 (t, J=5.9 Hz, 2H), 3.58 (t, J=5.2 Hz, 4H), 2.73 (t,J=5.4 Hz, 2H), 2.70 (t, J=5.9 Hz, 2H) 2.68 (t, J=5.2 Hz, 4H). The amineand hydroxyl protons were not observed. ¹³C NMR: δ 173.3, 151.7, 149.4,130.5, 129.5, 124.0, 123.4, 118.4, 63.5, 60.1, 57.3, 54.0, 46.6, 35.8.

Step C: β-Alanine, N-(Acridin-9-yl), 2-[bis(2-Chloroethyl)amino]ethylEster Dihydrochloride

SOCl₂ (0.5 ml) was added to a stirred suspension of β-alanine,N-(acridin-9-yl), 2-[bis(2-hydroxyethyl)amino]ethyl esterdihydrochloride from Step B (113 mg, 0.24 mmol) in CH₃CN (0.5 mL). Theresultant yellow solution was stirred at 23° C. for 16 h followed byremoval of the volatiles in vacuo. The remaining orange oil wasdissolved in EtOH (2 mL) and the EtOH was removed in vacuo to give ayellow solid. The material was then triturated with hexane (2×3 mL).Removal of residual solvents in vacuo gave 123 mg of the desiredmaterial, β-alanine, N-(acridin-9-yl), 2-[bis(2-chloroethyl)amino]ethylester dihydrochloride, (93% pure by HPLC) as a yellow solid. ¹H NMR: δ8.09 (apparent t, J=8.8 Hz, 4H), 7.66 (apparent t, J=7.6 Hz, 2H), 7.38(apparent t, J=7.7 Hz, 2H), 4.14 (t, J=5.9 Hz, 2H), 4.00 (t, J=5.8 Hz,2H), 3.43 (t, J=6.9 Hz, 4H), 2.87 (t, J=6.9 Hz, 4H), 2.77 (t, J=5.9 Hz,2H), 2.69 (t, J=5.8 Hz, 2H). The amine proton was not observed. ¹³C NMR:δ 173.0, 151.5, 149.4, 130.5, 129.6, 124.1, 123.4, 118.6, 63.5, 57.3,53.5, 46.7, 42.5, 35.7. IR (KBr pellet of HCl salt): 3423, 3236, 2939,2879, 1736, 1634, 1586, 1572, 1540, 1473, 1272, 1173 cm⁻¹.

Example 4 β-Alanine, N-(4-Methoxy-acridin-9-yl),2-[bis(2-Chloroethyl)amino]ethyl Ester Dihydrochloride, Compound IX

β-alanine, N-(4-methoxy-acridin-9-yl), methyl ester was prepared bymixing 1.4 g (5.84 mmol) of 4,9-dimethoxyacridine, 0.89 g (6.42 mmol) ofβ-alanine methyl ester hydrochloride and 20 ml of methanol and thenheating to reflux for 12 h under N₂. The reaction was then concentratedin vacuo, dissolved in CHCl₃-isopropanol (50 ml, 4:1 v/v), and washedwith 50% NH₄OH (2×5 ml) and brine (1×5 ml). The organic layer was driedwith Na₂SO₄ and concentrated in vacuo to yield 1.24 g (68% ) of themethyl ester (>74% purity by HPLC) as a yellow oil; R_(f)(SiO2, ethylacetate)=0.25; IR (thin film): 3363, 2947, 1730, 1611, 1573, 1518, 1484,1463, 1423, 1420, 1246, 1170, 1081 cm⁻¹; ¹H NMR: δ 2.70 (t, 2H, J=5.7Hz), 3.74 (s, 3H), 4.00 (t, 2H, J=6.3 Hz), 4.11 (s, 3H), 6.98 (d, 1H,J=7.4 Hz), 7.36 (m, 2H), 7.65 (m, 2H), 8.12 (d, 2H, J=8.5 Hz); ¹³C NMR):δ 35.7, 46.9, 52.3, 56.5, 107.2, 115.3, 119.8, 123.5, 124.1, 130.0,151.4, 173.6.

This was converted to the diol under conditions described in Example 3,Step B to afford 647 mg of a yellow oil. HPLC analysis of the crudemixture indicated a yield of 85% (λ=278 nm); R_(f)(SiO2, 20%methanol-ethyl acetate)=0.17; IR (thin film): 3337, 2947, 2828, 1726,1616, 1569, 1522, 1484, 1463, 1420, 1348, 1250, 1174, 1127, 1081, 1043cm⁻¹; ¹H NMR: δ 2.7 (m, 8H), 3.55 (m, 4H), 3.97-4.08 (m, 2H), 4.08 (s,3H), 4.19 (t, 2H, J=5.5 Hz), 6.96 (d, 1H, J=7.4 Hz), 7.29 (m, 2H), 7.61(m, 2H), 8.10 (m, 2H); ¹³C NMR: δ 36.0, 46.9, 53.7, 56.4, 57.1, 60.1,63.3, 107.4, 115.7, 119.1, 119.6, 123.2, 123.5, 123.9, 128.5, 130.0,140.8, 147.4, 151.6, 151.7, 154.3, 173.3.

This was converted to β-alanine, N-(4-methoxy-acridin-9-yl),2-[bis(2-chloroethyl)amino]ethyl ester dihydrochloride with thionylchloride as described in Example 3, Step C. Flash filtration (SiO₂) ofthe crude product using ethyl acetate followed by 10% methanol-ethylacetate gave 58 mg of a yellow oil after apparent on-column degradationof some product; R_(f)(SiO2, ethyl acetate)=0.26; IR (thin film): 3405,2955, 2828, 1726, 1616, 1577, 1518, 1463, 1416, 1348, 1246, 1174, 1123,1081, 1013 cm⁻¹; ¹H NMR: δ 2.69-2.99 (m, 8H), 3.45 (t, 4H, J=6.7 Hz),4.03 (m, 2H), 4.09 (s, 3H), 4.16 (t, 2H, J=5.9 Hz), 6.97 (d, 1H, J=7.7Hz), 7.32 (m, 2H), 7.65 (m, 2H), 8.12 (d, 2H, J=8.7 Hz).

The dihydrochloride salt could be isolated in crude form byconcentrating the reaction in vacuo with azeotropic removal of excessthionyl chloride (2×5 ml toluene). HPLC analysis indicated completeconsumption of the starting material and 4-methoxy acridone (R_(T)=22.3min) to be the major impurity. ¹H NMR (CD₃OD): δ 3.18 (t, 2H, J=6.4 Hz),3.71 (m, 6H), 4.04 (m, 4H), 4.18 (s, 3H), 4.51 (m, 2H), 7.17 (m, 2H),7.56 (m, 2H), 7.91-8.15 (m, 2H), 8.55 (d, 1H, J=8.8 Hz).

Similarly prepared from 3-chloro-9-methoxy-4-methylacridine wasβ-alanine, N-(3-chloro-4-methylacridin-9-yl),2-[bis(2-chloroethyl)amino]ethyl ester dihydrochloride, Compound X. ¹HNMR of the free base: δ 7.96-8.17 (m, 3H), 7.29-7.52 (m, 3H), 4.19 (t,J=5.8 Hz, 2H), 4.00 (s, 3H), 3.89 (t, J=5.1 Hz, 2H), 3.47 (t, J=6.8 Hz,4H), 2.91 (t, J=6.8 Hz, 4H), 2.83 (t, J=5.8 Hz, 2H), 2.67 (t, J=5.5 Hz,2H).

Similarly prepared from 6-chloro-2,9-dimethoxyacridine was η-alanine,N-(6-chloro2-methoxyacridin-9-yl), 2-[bis(2-chloroethyl)amino]ethylester dihydrochloride, Compound XIII. ¹H NMR of the free base: δ7.96-8.17 (m, 3H), 7.29-7.52 (m, 3H), 4.19 (t, J=5.8 Hz, 2H), 4.00 (s,3H), 3.89 (t, J=5.1 Hz, 2H), 3.47 (t, J=6.8 Hz, 4H), 2.91 (t, J=6.8 Hz,4H), 2.83 (t, J=5.8 Hz, 2H), 2.67 (t, J=5.5 Hz, 2H).

Example 5 β-Alanine, [N,N-bis(2-Chloroethyl)],3-[(6-Chloro-2-methoxyacridin-9-yl)amino]propyl Ester Dihydrochloride,Compound XI

Step A β-Alanine, [N,N-bis(2-Triisopropylsilyloxy)ethyl]ethyl Ester

A slurry of β-alanine ethyl ester hydrochloride (1.99 g, 12.9 mmol),K₂CO₃ (6.0 g, 43.4 mmol) and iodoethyl triisopropylsilyl ether (9.47 g,28.9 mmol) in acetonitrile (175 mL) were refluxed for 5-7 days. Aftervacuum evaporation of the solvent, the solid was triturated with CH₂Cl₂.The organic layer was washed with dilute Na₂CO₃(aq), then with brine anddried over anhydrous Na₂SO₄. The crude product was purified by silicagel chromatography (1:4 EtOAc/hexane) to provide 5.60 g of the oil,β-alanine, [N,N-bis(2triisopropylsilyloxy)ethyl]ethyl ester, (83.1% ).¹H NMR: δ 4.12 (q, J=7.1 Hz, 2H), 3.73 (t, J=6.8 Hz, 4H), 2.92 (t, J=7.3Hz, 2H), 2.70 (t, J=6.6 Hz, 4H), 2.46 (t, J=7.4 Hz, 2H), 1.4-0.9 (m,45H, includes triplet at 1.25 (3H) and singlets at 1.06 and 1.05).

Step B β-Alanine, N,N-bis(2-Triisopropylsilyloxy)ethyl

The β-alanine, [N,N-bis(2-triisopropylsilyloxy)-ethyl]ethyl ester fromStep A above (5.60 g, 10.8 mmol) and lithium hydroxide (0.59 g, 14.1mmol) were stirred in ethanol and refluxed for 3 h. The solvent wasremoved and the crude product was partitioned between CH₂Cl₂ and diluteNaHCO₃(aq). The organic layer was washed with brine, dried overanhydrous Na₂SO₄, and stripped to give β-alanine,N,N-bis(2-triisopropylsilyloxy)ethyl as a pale yellow oil (5.03 g, 95.1%yield). ¹H NMR: δ 3.90 (t, J=5.5 Hz, 4H), 3.04 (t, J=6.2 Hz, 2H), 2.92(t, J=5.5 Hz, 4H), 2.50 (t, J=6.1 Hz, 2H), 1.06 (s, 42H).

Step C: β-Alanine, [N,N-bis(2-Hydroxyethyl)],3-[(6-Chloro-2-methoxyacridin-9-yl)amino]propyl Ester

The β-alanine, N,N-bis(2-triisopropylsilyloxy)ethyl from Step B above(51.0 mg, 0.104 mmol) was stirred under N₂ in CH₂Cl₂ (1 mL). Whilechilling on an ice bath, SOCl₂ (0.5 mL) was added dropwise and thereaction was stirred for 2.25 h. After stripping the reaction mixture toremove excess SOCl₂, dry CH₂Cl₂ (0.5 mL) was added and the solution waschilled in an ice bath while under N₂. A chilled slurry of9-(3-hydroxy)propylamino-6chloro-2-methoxy-acridine (29.0 mg, 91.5 mmol)in CH₂Cl₂ (1 mL) was added. After 0.5 h the mixture was partitionedbetween CH₂Cl₂ and aqueous NaHCO₃. The organic layer was washed withbrine, dried with anhydrous Na₂SO₄, and stripped. The gum obtained wastriturated with hexane and the hexane extract was stripped to obtain avery crude mixture (53.5 mg) of triisopropylsilyl protected startingmaterial and product.

To remove the triisopropylsilyl groups, a portion of the crude protecteddiol (33.1 mg) was stirred in ice cold THF (1 mL). After the addition ofHF/pyridine (0.5 mL) the mixture was stirred at ambient temperatureunder a N2 filled balloon for 2.5 h. The reaction mix was partitionedbetween CH₂Cl₂ and NaHCO₃(aq) and the organic layer was washed severaltimes with dilute NaHCO₃(aq) to remove excess HF/pyridine. Afterpreliminary drying with brine, then with anhydrous Na₂SO₄, the solventwas stripped off to give crude diol (13.1 mg).

This was combined with additional crude diol (5.0 mg) and purified byC-18 preparative TLC with 95 CH₂Cl₂/5 iPA/1 TFA as eluent to obtain thediol TFA salt. After partitioning the salt between CH₂Cl₂ andNaHCO₃(aq), the organic layer was dried with brine, then with anhydrousNa₂SO₄, and stripped to give the free base of the diol, β-alanine,[N,N-bis(2-hydroxyethyl)],3-[(6-chloro-2-methoxyacridin-9-yl)amino]propyl ester, (5.0 mg). ¹H NMR:δ 7.92-8.25 (m, 3H), 7.23-7.47 (m, 3H), 4.30 (t, J=5.7 Hz, 2H), 3.98 (s,3H), 3.81 (t, J=6.2 Hz, 2H), 3.64 (t, J=4.9 Hz, 4H), 2.86 (t, J=6.1 Hz,2H), 2.67 (t, J=4.9 Hz, 4H), 2.51 (t, J=5.9 Hz, 2H), 2.04 (apparentquintet, 2H).

Step D: β-Alanine, [N,N-bis(2-Chloroethyl)],3-[(6-Chloro-2-methoxyacridin-9-yl)amino]propyl Ester Dihydrochloride,Compound XI.

The β-alanine, [N,N-bis(2-hydroxyethyl)],3-[(6-chloro-2-methoxyacridin-9-yl)amino]propyl ester from above (4.0mg, 0.0073 mmol) was dissolved in CH₂Cl₂ (1 mL) and chilled in anice/water bath. Ice cold SOCl₂ (0.1 mL) was added and the reaction wasallowed to stir for 4 h at room temperature. The reaction mixture wasstripped to remove solvent, triturated with hexane, and partitionedbetween CH₂Cl₂ and NaHCO₃(aq). After the organic layer was dried withbrine, then with anhydrous Na₂SO₄ and stripped, the dichlorocompound wasobtained as a yellow gum. ¹H NMR: δ 7.8-8.2 (m, 3H), 7.2-7.5 (m, 3H),4.35 (t, J=5.9 Hz, 2H), 3.85-4.10 (3.99, s, OMe and 3.9-4.0, m, NHCH ₂,total 5H), 3.48 (t, J=6.9 Hz, 4H), 2.9-3.0 (m, 6H), 2.49 (t, J=6.6 Hz,2H), 2.1-2.3 (m, 2H).

The free amine was stirred in chilled CH₂Cl₂, acidified with 1M HCl inether and stripped with a few drops of methanol to obtain the desiredcompound, β-alanine, [N,N-bis(2-chloroethyl)],3-[(6-chloro-2-methoxyacridin-9-yl)amino]propyl ester dihydrochloride(2.5 mg), (3.5 mg, 81%), as a yellow solid.

In the same manner as given in the foregoing Step C, but using6-chloro-9-(2-hydroxy)ethylamino-2-methoxy-acridine instead of6-chloro-9-(3-hydroxy)propylamino-2 methoxy-acridine, was prepared theanalogous diol. ¹H NMR: δ 7.96-8.13 (m, 3H), 7.20-7.47 (m, 3H), 4.76 (t,J=4.9 Hz, 2H), 3.99 (s, 3H), 3.92-4.14 (m, 2H), 3.60 (t, J=5.1 Hz, 4H),2.78 (t, J=6.1 Hz, 2H), 2.63 (t, J=5.1 Hz, 4H), 2.45 (t, J=6.0 Hz, 2H).By analogy to Step D this was converted to β-alanine,[N,N-bis(2-chloroethyl)], 2-[(6-chloro-2 methoxyacridin-9-yl)amino]ethylester dihydrochloride, Compound XII. ¹H NMR: δ 7.94-8.20 (m), 7.20-7.50(m), 4.42 (CH ₂OC═O), 3.90-4.10 (OCH ₃, NHCH ₂), 3.46 (CH ₂Cl), 2.82(N(CH ₂)₃), 2.39-2.56 (CH ₂C═O).

Example 6 [N,N-bis(2-Chloroethyl)]-2-aminoethyl4,5′,8-Trimethyl-4′-psoralenacetate Hydrochloride, Compound XIV

Step A: [N,N-Bis(2-Hydroxyethyl)]-2-aminoethyl4,5′8-Trimethyl-4′-psoralenacetate

A slurry of methyl 4,5′8-trimethyl-4′-psoralenacetate (250 mg, 0.832mmol), triethanolamine (12 mL) and 1M HCl in ether (2 mL) were stirredat 100° C. for 2 h. The resulting clear brown solution was allowed tocool to room temperature and partitioned between CH₂Cl₂ and saturatedNaHCO₃(aq). The organic layer was rinsed several times with saturatedNaHCO₃(aq). After drying with anhydrous Na₂SO₄, solvent was removed invacuo and the residue was partitioned between CH₂Cl₂ and 1M aq. HCl. Theaqueous layer was rinsed several times with CH₂Cl₂ and then made basicwith K₂CO₃ (s) in the presence of the organic solvent. The organic layercontaining the neutral product was rinsed with water several times, thendried and concentrated. A repetition of the acid-base extractionprocedure gave the desired product as a beige solid (84.3 mg, 24.3% ):¹H NMR: δ 7.53 (s, 1H), 6.24 (s, 1H), 4.23 (t, J=5.4 Hz, 2H), 3.69 (s,2H), 3.56 (t, J=5.3 Hz, 4H), 2.82 (t, J=5.4 Hz, 2H), 2.69 (t, J=5.3 Hz,4H), 2.57 (s, 3H), 2.51 (d, J=1.1 Hz, 3H), 2.47 (s, 3H).

Step B: [N,N-bis(2-Chloroethyl)]-2-aminoethyl4,5′8-Trimethyl-4′-psoralenacetate Hydrochloride

Thionyl chloride (0.2 mL) was added to an ice cold mixture of the abovediol (9.8 mg, 0.023 mmol) in CH₂Cl₂ (1 mL) and stirred at roomtemperature overnight under nitrogen. The resulting slurry wasconcentrated then triturated with hexane to give the desired product(6.2 mg, 53.9%) as an off-white solid: ¹H NMR (CD₃OD): δ 7.71 (s, 1H),6.28 (s, 1H), 4.56 (t, J=4.8 Hz, 2H), 3.95 (t, J=6.1 Hz, 4H), 3.89 (s,2H), 3.60-3.83 (m, 6H), 2.54 (s, 3H), 2.53 (s, 3H), 2.50 (s, 3H).

Example 7 Synthesis of β-Alanine, N-(Acridin-9-yl),2-[bis(2-Chloroethyl)amino]ethyl Amide (Compound XV)

Step A2-[N′,N′-bis(2-hydroxyethyl)]-N-(tert-Butoxycarbonyl)ethylenediamine

To a solution of N-(tert-butoxycarbonyl)ethanolamine (1.21 g, 7.5 mmol)and triethylamine (1.57 mL, 1.1 g, 11 mmol) in dry CH₂Cl₂ (25 mL) at 0°C. was added methanesulfonyl chloride (0.64 mL, 0.95 g, 8.3 mmol)dropwise. The reaction was stirred at 0° C. for 1 h, allowed to warm to23° C. and was stirred overnight. The volatiles were removed in vacuo togive the mesylate as a white solid. Diethanolamine (7.2 mL, 7.9 g, 75mmol) was added and the reaction mixture was heated to 75° C. withstirring for 6 h. The crude reaction mixture was partitioned between H₂O(60 mL) and CHCl₃ (4×20 mL). The combined organic layers were washedwith brine (20 mL) and dried over Na₂SO₄. Removal of solvent in vacuogave 1.21 g (65% ) of the diol as a thick-yellow oil, ¹H NMR: δ5.51-5.39 (m, 1H), 3.61 (t, J=4.9 Hz, 4H), 3.29-3.13 (m, 2H), 2.68-2.52(m, 6H), 1-44 (s, 9H). The hydroxyl protons were not observed,

Step B2-[N′,N′-bis(2-tert-Butyldimethylsilyloxyethyl)]-N-(tert-butoxycarbonyl)ethylenediamine

To a stirred solution of the diol from step A (1.21 g, 4.87 mmol) andpyridine (1.59 mL, 1.55 g, 19.6 mmol) in dry CH₂CL₂ (12 mL) at 0° C. wasadded tert-butyldimethylsilyl chloride (2.21 g, 14.7 mmol). The reactionmixture was allowed to warm to 23° C. and was stirred for 2 d. Thereaction mixture was diluted with CH₂Cl₂ (80 mL) and washed with H₂O(3×25 mL) and then brine (3×25 mL). The organic layer was dried overNa₂SO₄. Removal of solvent in vacuo) gave 2.26 g (97% ) of a pale yellowoil. ¹H NMR: δ 5.37-5.22 (m, 1H), 3.62 (t, J=6.2 Hz, 4H), 3.19-3.08 (m,2H), 2.63 (t, J=6.2 Hz, 6H), 1.42 (s, 9H), 0.873 (s, 18H), 0.04 (s,12H).

Step C: 2-[N,N,bis(2,tert-Butyldimethylsilyloxyethyl)]ethylenediamine

To a flask containing the protected amine from step B (4.24 g, 8.89mmol) was added 5 mL of trifluoroacetic acid at 23° C. The reactionmixture was stirred for 15 minutes at 23° C. followed by removal of thetrifluoracetic acid in vacuo. The crude product was partitioned between2 N NaOH (100 mL) and CH₂Cl₂ (3×35 mL). The combined organic layers weredried over Na₂SO₄. Removal of solvent in vacuo gave 1.76 g (53% ) of ayellow oil. ¹H NMR: δ 3.66 (t, J=6.5 Hz, 4H), 2.72-2.53 (m, 8H),1.72-1.63 (m, 2H), 0.87 (s, 18H), 0.02 (s, 12H).

Step D: β-Alanine, N-(tert-Butoxycarbonyl),2-[bis(2-Tertbutyldimethylsilyloxyethyl)amino]ethyl Amide

To a solution of 3-(N-tert-butoxycarbonyl)aminopropanoic acid (822.0 mg,4.34 mmol) and 4-methylmorpholine (442.0 mg, 4.37 mmol) in 14 mL of dryTHF at −15° C. was added isobutylchloroformate (0.53 mL, 0.56 g, 4.1mmol). The reaction mixture was stirred at −15° C. for 1 min followed bythe addition of the amine from step C (1.72 g, 4.57 mmol). The reactionmixture was allowed to warm to 23° C. and was stirred for 1 h. Themixture was then filtered, the precipitate was washed with THF (5 mL)and the filtrate was concentrated in vacuo. The remaining material waspartitioned between 2 N NaOH (50 mL) and CH₂Cl₂ (3×20 mL). The combinedorganic layers were dried over Na₂SO₄ and the solvent removed in vocuoto give 2.25 g of a brownish yellow gum. Purification of the crudematerial (2.25 g) by medium pressure liquid chromatography (silica gel,1:1 CHCl₃/EtOAc) gave 627.0 mg (26% ) of a pale yellow oil. ¹H NMR: δ3.63 (t, J=6.2 Hz, 4H), 3.54-3.35 (m, 4H), 3.20-3.19 (m, 2H), 2.71-2.50(m, 6H), 1.43 (s, 9H), 0.89 (s, 18H), 0.05 (s, 12H). The amide andcarbamate protons were not observed.

Step E: β-Alanine, 2-(bis(2-tert-Butyldimethylsilyloxyethyl)amino]ethylAmide

The protected amine formed in step D (627.0 mg, 1.14 mmol) was dissolvedin trifluoroacetic acid (5 mL) at 23° C. The resulting solution wasstirred for 5 min (until CO₂ evolution ceased) followed by removal ofthe trifluoroacetic acid in vacuo. The remaining material waspartitioned between saturated NaHCO₃ (50 mL) and CH₂Cl₂ (3×20 mL). Thecombined organic layers were dried over Na₂SO₄ and the solvent removedin vacuo to give 203.4 mg (40% ) of a pale yellow oil.

Step F: β-Alanine, N-(Acridin-9-yl),2-[bis(2-tert-Butyldimethylsilyloxyethyl)amino]ethyl Amide

A mixture of the crude amine from step E (203.4 mg, 0.45 mmol),9-methoxyacridine (96.8 mg, 0.46 mmol) and methanol (10 mL) was heatedto reflux for 4 h. The reaction mixture was allowed to cool to 23° C.and was stirred for an additional 2.5 days. The methanol was removed invacuo and the remaining material was partitioned between 2N NaOH (50 mL)and CH₂Cl₂ (3×20 mL). The combined organic layers were dried over Na₂SO₄and the solvent removed in vacuo to give 69.6 mg of a yellow oil.Purification of the crude material (69.6 mg) by TLC (silica gel, 1:1CHCl₃/EtOAC) gave 23.4 mg (8.3% ) of a yellow oil. ¹H NMR: δ 8.19 (d,J=8.8 Hz, 2H), 8.06 (d, J=8.8 Hz, 2H), 7.65 (br t, J=7.6 Hz, 2H), 7.36(br t, J=7.6 Hz, 2H), 6.8-6.7 (m, 1H), 4.06 (t, J=5.6 Hz, 2H), 3.61 (t,J=5.8 Hz, 4H), 3.373.32 (m, 2H), 2.72-2.61 (m, 6H), 2.51 (t, J=5.5 Hz,2H), 0.86 (s, 18H), 0.02 (s, 12H). The amine proton was not observed,¹³CNMR: δ 172.1, 152.4, 149.3, 130.5, 129.3, 123.7, 118.0, 112.8, 62.3,57.4, 54.1, 47,4, 38.2, 36.5, 26.4, 18.8, −4.8.

Step G: β-Alanine, N-(Acridin-9-yl), 2-[bis(2-Hydroxyethyl)amino]ethylAmide Dihydrochloride

To a stirred solution of the bis-protected diol from step F (22.0 mg,0.04 mmol) in isopropanol (1.0 mL) was added a 5-6 N HCl/isopropanolsolution (0.05 mL) at 23° C. The reaction mixture was stirred at 23° C.for 17 h and the resultant yellow precipitate was collected by vacuumfiltration. The yellow solid was rinsed with an additional 1.0 mL ofisopropanol. Residual isopropanol was removed in vacuo (overnight) togive 11.4 mg (69% ) of the diol dihydrochloride salt as a yellow solid.¹H NMR (CD₃OD): δ 8.52 (d, J=8.8 Hz, 2H), 7.96 (br t, J=7.5 Hz, 2H),7.82 (d, J=8.4 Hz, 2H), 7.57 (br t, J=7.5 Hz, 2H), 4.49 (t, J=6.2 Hz,2H), 3.91 (t, J=4.8 Hz, 4H), 3.74-3.56 (m, 2H), 3.53-3.38 (m, 6H), 2.97(t, J 6.1 Hz, 2H). The amide, amine and hydroxyl protons were notobserved.

Step H: β-Alanine, N-(Acridin-9-yl), 2-[bis(2-Chloroethyl)amino]ethylAmide Dihydrochloride

To a stirred suspension of the diol from step G (11.4 mg, 0.024 mmol) inCH₃CN (1.0 mL) was added SOCl₂ (0.12 mL, 200 mg, 1.7 mmol) at 23° C. Thereaction mixture was stirred at 23° C. for 15 minutes and the solutionwas heated to 50° C. for 3.5 h. The resultant yellow precipitate wascollected via vacuum filtration and was rinsed with CH₃CN (3×1.0 mL) anddried in vacuo to give 8.3 mg (67% ) of a yellow powder (95% pure byHPLC). ¹H NMR (CD₃OD): δ 8.55 (d, J=8.7 Hz, 2H), 8.00(br t, J=7.7 Hz,2H), 7.84 (d, J=8.7 Hz, 2H), 7.59 (br t, J=7.7 Hz, 2H), 4.51 (t, J=6.2Hz, 2H), 3.98 (t, J=5.7 Hz, 4H), 3.71 (t, J=5,7 Hz, 4H), 3.65-3.55 (m,2H), 3.55-3.42 (m, 2H), 2.99 (t, J=6.2 Hz, 2H). The amide and amineprotons were not observed.

Example 8 Hydrolysis of the Frangible Compounds

For the frangible compounds incorporating an ester group (“forward” and“reverse” esters) in the frangible linker, model compounds were studiedto determine the amount of ester hydrolysis.

The reaction

AcrNH—(CH₂)_(n)—C(═O)—OR→AcrNH—(CH₂)_(n)—CO₂H+HO—R

(“forward ester”) (“acridine acid”)

where AcrNH indicates a 9-amino acridine bearing substituents asindicated in the following table, and n and R are as indicated, wasstudied. Table III shows the rate enhancement for ester hydrolysis whenthe ester linkage is situated between, and in proximity to, an acridinering and an alkylamino group. The hydrolysis rate is rapid regardless ofwhether the acridine moiety is positioned at the acid terminus of theester, or at the alcohol terminus.

TABLE III Percent Hydrolysis at 100 minutes (aqueous solution, pH 8, 37°C.) Acridine R = R = R = Substituent(s) n = methyl —(CH₂)₂N(CH₂CH₂OH)₂—(CH₂)₃N(CH₂CH₂OH)₂ 6-Cl, 2-OMe 1 28%  6-Cl, 2-OMe 2 22%  6-Cl, 2-OMe 35% 6-Cl, 2-OMe 4 2% 6-Cl, 2-OMe 7 <1%   2-CO₂CH₃ 2 9% 55% 57% 2-CO₂CH₃ 318% 2-CO₂CH₃ 4 17%

For the reaction

AcrNH—(CH₂)_(n)—O—C(═O)—R→AcrNH—(CH₂)_(n)—OH+HOC(═O)—R

(“reverse ester”) (“acridine alcohol”)

where AcrNH indicates a 9-amino acridine bearing substituents asindicated in the following table, and n and R are as indicated, thefollowing results were obtained:

TABLE IV Percent Hydrolysis at 100 minutes (aqueous solution, pH 8, 37°C.) Acridine R = R = R = Substituent(s) n = methyl —(CH₂)₂N(CH₂CH₂OH)₂—(CH₂)₃N(CH₂CH₂OH)₂ 6-Cl, 2-OMe 2 2% 99% 6-Cl, 2-OMe 3 2% 65%

At pH 3, all compounds in Tables III and IV showed ≦1% hydrolysis at 100min.

The mustard compounds cannot be evaluated in the same manner sincemultiple degradation pathways occur simultaneously. Nevertheless, whenCompound VIII is incubated under the same conditions as in Tables IIIand IV, the acridine acid is the major product (≧95% ) after longincubation times and 40% is formed at 100 minutes. This comparesfavorably to the table entry for the analogous diol (57% hydrolysis at100 minutes).

It will be appreciated from the data in Tables III and IV that thehydrolysis rate of the ester linkage varies inversely with the length ofthe linker arm between the 9-aminoacridine moiety and the ester group(in Tables III and IV, as n increases, the amount of hydrolysis at 100minutes decreases). This provides a method of tuning the hydrolysis rateof the compounds. This ability to tune the breakdown of the linkerallows compound reactivity to be adjusted for various applications, asdesired.

MATERIALS

The following materials were used in the following Examples:

While it is commercially available from Baxter Healthcare Corp.,Deerfield, Ill., Adsol used in this and the following experiments wasmade by sterile filtering the following mixture: 22 g glucose, 9 g NaCl,7.5 g mannitol, and 0.27 g adenine in 1 liter of distilled water.

Quinacrine mustard was obtained from Aldrich Chemical Co., St. Louis,Mo.

Whole blood was obtained from the Sacramento Blood Center (SacramentoCalif.).

Example 9 Inactivation of Vesicular Stomatitis Virus (VSV)

Stock solutions (typically 10-30 mM) of each compound are prepared bydissolving an appropriate amount of material in blood bank salinepreviously acidified with 2 mM H₃PO₄, then quickly frozen in 1 mLaliquots. At the time of use, aliquots are warmed to ≦10° C. and usedwithin one hour.

For preparation of packed red blood cells (PRBC), whole blood, withmeasured Hct, is centrifuged at 3800 rpm for 6 min. Supernatant plasmais removed and measured. Adsol is added to provide PRBC with 60% Hct.Plasma concentration is 15-20% .

A VSV (stock solution, approx. 10⁹ pfu/mL, obtained from ATCC AmericanType Cell Culture, Rockville, Md.) is diluted 1:10 into tissue culturemedium (DMEM with 10% NCS) or into PBRC to provide the test medium whichis aliquoted (1 mL) into 2 mL sterile o-ring tubes.

To each tube is added sufficient test compound solution to provide atest compound concentration of 10-300 μM. Each sample is quickly mixedby fully pipetting the mixture several times. Suspensions are incubatedat ambient temperature for 4 h. Virus titer was ascertained followingincubation of the treated medium in BHK (baby hamster kidney) hostcells. PRBC was used directly rather than the supernatant alone. Viruskill was inversely proportional to the appearance of plaques in the cellcultures. The difference between the titer of the untreated test mediumand that of a treated sample provides the log kill for the compound atthat concentration. The detection limit is 10¹⁴ pfu/mL.

In tissue culture medium, quinacrine mustard (QM), and Compounds IV, VI,XI, VII, and VIII inactivated >3 logs of VSV at <50 μM test compound.Compound XII inactivated 2 logs at approx. 200 μM. This compound isbelieved to be particularly unstable with respect to ester hydrolysis.As indicated in the first entry in Example 8, Table IV, thecorresponding diol compound (β-alanine, [N,N-bis(2-hydroxyethyl)],2-[(6-chloro-2-methoxyacridin-9-yl)amino]ethyl ester) was 99% hydrolyzedafter 100 minutes at pH 8, 37° C. It is likely that the mustard compoundalso underwent rapid hydrolysis. This illustrates the importance of theanchor moiety for directing the effector portion of the molecule tonucleic acid, and the importance of tuning the reactivity of the9-aminoacridine class of compounds so that they are effective underconditions of actual use. Under the described inactivation protocol,hydrolysis of Compound XII is expected to be competitive withinactivation.

In PRBC, QM and Compounds IV, VI, VIII, V, and XIII inactivated >2 logsof VSV at <150 μM of test compound.

Example 10 Inactivation of Yersinia enterocolitica

PRBC and stock solutions were prepared as for VSV in Example 9. Yersinia(California Department of Health Services, Microbial Disease Laboratory,Berkeley, Calif.) is cultured in LB-broth at 37° C. overnight on ashaker. A portion (10 mL) is centrifuged at 2500 rpm for 10 min in a 15mL conical tube. The pellet is resuspended in 1 mL of Adsol to provideapprox. 10⁹ bacteria/mL. To measure the titer, the optical density ismeasured of a 1:100 dilution in Adsol (OD₆₁₀=0.2 at 10⁸ bacterial/mL).The bacterial stock is then diluted 1:100 into saline or PRBC to providethe test medium which is aliquoted (1 mL) into 2 mL o-ring steriletubes.

To each tube is added a sufficient amount of the test compound solutionto provide a test compound concentration of 10-300 μM. Each sample isquickly mixed by fully pipetting the mixture several times. It is thenincubated for two hours at ambient temperature, then plated out onLB-agar starting with 100 μL sample starting at 10⁻¹ dilution andcontinuing dilutions to 10⁻⁸. The plates are incubated overnight at 37°C. and the colonies are counted. The difference between the titer of theuntreated test medium and that of a treated sample provides the log killfor the compound at that concentration. The detection limit is 10bacteria/mL.

In saline, quinacrine mustard (QM) and Compounds IV, VI, VIII, V, IX,and X inactivated >2 logs of Yersinia at concentrations ≦200 μM.

In PRBC, QM and Compounds VI, VIII, V, X, and XIII inactivated >2 logsof Yersinia at concentrations ≦200 μM.

Example 11 Blood Function Assay After Introduction of a Compound of theInvention

One of the contemplated uses of the compounds of the invention involvesthe introduction of one or more of the compounds of the invention intoblood or blood products intended for transfusion. The blood or bloodproduct must remain suitable for transfusion after treatment with thecompounds. To evaluate the effect of the compounds on red blood cellfunction, the compounds were tested as described below.

Packed red blood cells with a 50% hematocrit (Hct) is prepared byspinning whole blood with a known Hct at 2500 rpm for 6 min. Thesupernate is removed and measured. The suspension is diluted with asufficient volume of Adsol to achieve the desired Hct. 1.5 mL of PRBC isplaced in each 2 ml o-ring tube and enough of the stock solution of thetest compound is added to achieve the desired concentration. The samplesare incubated at ambient temperature for 4 hours, then stored overnightat 4° C. Hemolysis was determined as described in Hogman et al.,Transfusion, 31:26-29 (1991).

A lysis standard is prepared for each sample by diluting 10 μM of theincubated mixture in two steps with water to give a final 1:4000dilution.

For the assay, samples were removed from 4° C. storage and warmed for<15 minutes. After vortexing briefly to mix, an aliquot was removed andspun for 2 minutes at 14,000 rpm. The supernate was removed and spun for10 minutes at 14,000 rpm. The supernate was removed and diluted asneeded in water. The absorbance at 414 nm of the lysis standards and thediluted supernates were read against a water blank. Percent hemolysiswas calculated as:

(100% −50% Hct)×(A₄₁₄ of sample×dilution factor)/(A₄₁₄ of lysisstandard×4000)

The A₄₁₄ of the samples is uncorrected for any absorbance due to thepresence of the compound of the invention. The results are given inTable Va.

TABLE Va Hemolysis Data at Day 1 Percent Compound and ConcentrationHemolysis Number of Samples Tested BBS* 0.066 14  ABBS** 0.065 8 150 μMQM*** 0.220 14  300 μM QM 0.320 14  150 μM   IV 0.091 14  300 μM   IV0.109 14  150 μM   VI 0.103 14  300 μM   VI 0.140 14  150 μM   VIII0.110 6 300 μM   VIII 0.135 6 150 μM   V 0.136 2 300 μM   V 0.149 2 150μM   IX 0.116 2 300 μM   IX 0.099 2 150 μM   X 0.121 2 300 μM   X 0.1532 *BBS = blood bank saline **ABBS = acidic blood bank saline ***QM =quinacrine mustard

Extracellular potassium was measured using a Ciba Corning Model 614K⁺/Na⁺ Analyzer (Ciba Coning Diagnostics Corp., Medford, Mass.). ATP wasmeasured using Sigma procedures No. 366 (Sigma, St. Louis, Mo.).

Table Vb. shows the relative values of extracellular potassium relativeto the control values of the untreated PRBC samples for that experiment.For example, a relative value of 1.03 meant that the treated sample has3% more extracellular potassium concentration than the untreatedcontrol.

TABLE Vb Relative Extracellular Potassium Levels (replicates)* Con- Com-centration pound (μM) Day 1 Day 7 Day 14 IV 100 1.01 (1) 0.98 (1) 1.03(1) 200 1.05 (1) 1.15 (1) 1.01 (1) 300 1.03 (1) 1.15 (1) 1.15 (1) V 3001.04-1.46 (4) 0.96-1.01 (4) 0.95-1.01 (4) *[K+] (treated)/[K+](untreated)

Table Vc. shows the relative values ATP relative to the control valuesof the untreated PRBC samples for that experiment. For example, arelative value of 1.03 meant that the treated sample has 3% more ATPthan the untreated control.

TABLE Vc Relative ATP Levels (replicates)* Con- Com- centration pound(μM) Day 1 Day 7 Day 14 IV 100 1.01 (1) 0.93 (1) 0.94 (1) 200 1.05 (1)0.94 (1) 0.94 (1) 300 1.03 (1) 0.93 (1) 0.92 (1) V 300 0.96-1.00 (4)0.91-1.01 (4) 0.94-1.01 (4) *[ATP] (treated)/[ATP] (untreated)

Example 12 Inactivation of HIV by Compounds of the Invention

Cell associated HIV in TC Medium (Popovic et al., Science, 224:497(1984): H9-IIIb cells are suspended in TC Medium to provide a suspensionwith a titer of approximately ≧10⁶ pfu/mL. To 2 mL aliquots of the testmedium in 15 mL conical tubes is added a sufficient amount of testcompound solution to achieve the desired concentration of activematerial. The suspensions are immediately mixed by fully pipettingseveral times, then vortexing briefly. The samples are incubated atambient temperature for 2-4 h, then centrifuged. The pellets areresuspended in 1 mL of plaque assay diluent, then quickly frozen at −80°C. and titrated by a microplaque assay. (Hanson et al., J. Clin. Micro.,28:2030 (1990)).

Compounds quinacrine mustard, IV and VI inactivated >3 logs of HIV at≦25 μM of test compound.

Cell-associated HIV in PRBC: For assays run in PRBC, the packed cellsare prepared as described in the VSV assay. The HIV9-IIIb cells areadded to the Adsol prior to dilution of the centrifuged cells. Theresultant suspension is mixed by fully pipetting all the material. Uponcompletion of incubation of the test compound, the samples are dilutedwith 3 mL of a 1:1 plasma:DMEM solution containing 5 μL of heparin. Theinfected cells are then isolated using a fycol-hypaque gradient,resuspended in 1 mL of the diluent, and frozen for later titration.

Compounds quinacrine mustard, VI and V inactivated >3 logs of HIV at≦200 μM of test compound.

Cell-free HIV in PRBC: The protocol is similar to that described above,except that cell-free HIV is added directly to the PRBC afterpreparation. After incubation, the medium is centrifuged and thesupernate is frozen for later titration.

Compounds quinacrine mustard, IV, V and VI inactivated >3 logs of HIV at<100 μM of test compound.

Although the forgoing invention has been described in some detail by wayof illustration and example for purposes of clarity and understanding,it will be apparent to those skilled in the art that certain changes andmodifications may be practical. Therefore, the description and examplesshould not be construed as limiting the scope of the invention, which isdelineated by the appended claims. The entirety of U.S. Pat. Nos.5,559,250 and 5,399,719 are hereby incorporated by reference. All otherpatents and references cited herein are hereby incorporated byreference.

What is claimed is:
 1. A compound having the formula:

where R₁, R₂, R₃, R₄, R₅, R₆, R₇, and R₈ are independently selected fromthe group consisting of —H, —R₁₀, —O—R₁₀, —NO₂, —NH₂, —NH—R₁₀, —N(R₁₀)₂,—F, —Cl, —Br, —I, —C(═O)—R₁₀, —C(═O)—O—R₁₀, and —O—C(═O)—R₁₀, where —R₁₀is independently H, —C₁₋₈alkyl, —C₁₋₈heteroalkyl, -aryl, -heteroaryl,—C₁₋₃alkyl-aryl, —C₁₋₃heteroalkyl-aryl, —C₁₋₃alkyl-heteroaryl,—C₁₋₃heteroalkyl-heteroaryl, -aryl-C₁₋₃alkyl, -aryl-C₁₋₃heteroalkyl,-heteroaryl-C₁₋₃alkyl, -heteroaryl-C₁₋₃heteroalkyl,—C₁₋₃alkyl-aryl-C₁₋₃alkyl, —C₁₋₃heteroalkyl-aryl-C₁₋₃alkyl,—C₁₋₃alkyl-heteroaryl-C₁₋₃alkyl, —C₁₋₃alkyl-aryl-C₁₋₃heteroalkyl,—C₁₋₃heteroalkyl-heteroaryl-C₁₋₃alkyl,—C₁₋₃heteroalkyl-aryl-C₁₋₃heteroalkyl,—C₁₋₃alkyl-heteroaryl-C₁₋₃heteroalkyl, or—C₁₋₃heteroalkyl-heteroaryl-C₁₋₃heteroalkyl; R₂₀ is —H or —CH₃; and R₂₁,is —R₁₁—W—X—E, where —R₁₁— is independently —C₁₋₈alkyl-,—C₁₋₈heteroalkyl-, -aryl-, -heteroaryl-, —C₁₋₃alkyl-aryl-,—C₁₋₃heteroalkyl-aryl-, —C₁₋₃alkyl-heteroaryl-,—C₁₋₃heteroalkyl-heteroaryl-, -aryl-C₁₋₃alkyl-, -aryl-C₁₋₃heteroalkyl-,-heteroaryl-C₁₋₃alkyl-, -heteroaryl-C₁₋₃heteroalkyl-,—C₁₋₃alkyl-aryl-C₁₋₃alkyl-, —C₁₋₃heteroalkyl-aryl-C₁₋₃alkyl-,—C₁₋₃alkyl-heteroaryl-C₁₋₃alkyl-, —C₁₋₃alkyl-aryl-C₁₋₃heteroalkyl-,—C₁₋₃heteroalkyl-heteroaryl-C₁₋₃alkyl-, —C₁₋₃heteroalkyl-aryl-C₁₋₃heteroalkyl-, —C₁₋₃alkyl-heteroaryl-C₁₋₃heteroalkyl-, or—C₁₋₃heteroalkyl-heteroaryl-C₁₋₃heteroalkyl-; W is independently—C(═O)—O—, —O—C(═O)—, —C(═S)—O—, —O—C(═S)—, —C(═S)—S—, —C—C(═S)—,—C(═O)—S—, —S—C(═O)—, —O—S(═O)₂—O—, —S(═O)₂—O—, —O—S(═O)₂—,—O—P(═O)(—OR₁₀)—O—, —O—P(═O)(—OR₁₀)—O—, —P(═O)(—OR₁₀)—O—,—O—P(═O)(—OR₁₀)—; X is independently —R₁₁—; and E is independentlyselected from the group consisting of —N(R₁₂)₂, —N(R₁₂)(R₁₃), —S—R₁₂,and

 where —R₁₂ is —CH₂CH₂—G, where each G is independently —Cl, —Br, —I,—O—S(═O)₂—CH₃, —O—S(═O)₂—CH₂—C₆H₅, or —O—S(═O)₂—C₆H₄—CH₃; and where R₁₃is independently —C₁₋₈alkyl, —C₁₋₈heteroalkyl, -aryl, -heteroaryl,—C₁₋₃alkyl-aryl, —C₁₋₃heteroalkyl-aryl, —C₁₋₃alkyl-heteroaryl,—C₁₋₃heteroalkyl-heteroaryl, -aryl-C₁₋₃alkyl, -aryl-C₁₋₃heteroalkyl,-heteroaryl-C₁₋₃alkyl, -heteroaryl-C₁₋₃heteroalkyl,—C₁₋₃alkyl-aryl-C₁₋₃alkyl, —C₁₋₃heteroalkyl-aryl-C₃alkyl,—C₁₋₃alkyl-heteroaryl-C₁₋₃alkyl, —C₁₋₃alkyl-aryl-C₁₋₃heteroalkyl,—C₁₋₃heteroalkyl-heteroaryl-C₁₋₃alkyl,—C₁₋₃heteroalkyl-aryl-C₁₋₃heteroalkyl,—C₁₋₃alkyl-heteroaryl-C₁₋₃heteroalkyl, or—C₁₋₃heteroalkyl-heteroaryl-C₁₋₃heteroalkyl; and all salts andstereoisomers [(including enantiomers and diastereomers)] thereof.
 2. Amethod of making a compound of claim 1, wherein the method comprises thestep of reacting an amino ester with a 9-substituted acridine to providean N-(9-acridinyl)amino ester.
 3. A method according to claim 2, whereinthe amino ester is an ω-amino alkanoic ester.
 4. A method according toclaim 2, wherein the amino ester is a β-alanine derivative.
 5. A methodaccording to claim 2, wherein the amino ester comprises a protecteddiol.
 6. A method according to claim 5, further comprising the step ofdeprotecting the protected diol substituent to form anN-(9-acridinyl)amino ester comprising a diol substituent.
 7. A methodaccording to claim 2, wherein the amino ester comprises a protectedbis(hydroxyalkyl)amine.
 8. A method according to claim 2, wherein the9-substituted acridine is a 9-methoxy acridine.
 9. A method according toclaim 2, wherein the 9-substituted acridine is a 9-chloro acridine. 10.A method according to claim 2, further comprising the step of convertingthe N-(9-acridinyl)amino ester into an N-(9-acridinyl)amino estercomprising a diol substituent.
 11. A method according to claim 10,wherein the step of the step of converting the N-(9-acridinyl)aminoester into an N-(9-acridinyl)amino ester comprising a diol substituentcomprises a transesterification reaction.
 12. A method according toclaim 10, wherein the method further comprises the step of replacing thehydroxyl groups of the diol substituent with chloro groups.